Hauck LabWiki - User contributions [en-gb]

AG Hauck - LabWiki

2025-06-16T12:14:00Z

ESO wikiadmin:

[[File:AG Hauck Group photo September 2023.jpg|thumb|Gruppenfoto - September 2023]]

== '''Welcome!''' ==
This is the '''LabWiki''' – your central reference for all methods, rules, and protocols in our laboratory!

This wiki is designed to help you work safely, efficiently, and reliably. Here you will find guides on various work techniques, important safety regulations, and documentation on equipment and materials.

The data and protocols have been curated and carefully prepared over a long period of time. Nevertheless, there may still be errors in a few areas. Please let us know in the discussion section of the respective article.

Knowledge grows when shared. That's why we appreciate it when you contribute your knowledge here and help us continue developing it.
 {{Box
|Titel= ⚠️ The most important thing you can find here?
|Inhalt=
'''[[Lab Etiquette|Lab Rules]]:''' Correct behaviour in the lab - from deadly sins to too much blotto. '''First port of call for every lab novice'''!
}}
{{Labor-Boxes}}

= 🖨️ How to Print Pages from the Wiki =

== Quick Print Access ==

=== Windows ===

* '''Keyboard shortcut:''' <code>Ctrl + P</code>
* '''Alternative:''' Right-click on page → "Print"
* '''Menu:''' Browser menu (⋮) → "Print"

=== Mac ===

* '''Keyboard shortcut:''' <code>⌘ + P</code> (Command + P)
* '''Alternative:''' Right-click on page → "Print"
* '''Menu:''' Safari/Chrome menu → "Print"

== Optimizing Print Settings ==

=== 📄 Save as PDF ===

# Open print dialog (<code>Ctrl + P</code> / <code>⌘ + P</code>)
# '''Destination:''' Select "Save as PDF"
# Click '''Save'''
# Choose filename and save location

=== 📏 Adjusting Scale ===
'''Problem:''' Text too small or cut off?

'''Solution:'''

* '''Scale:''' 100% (default) → Reduce to 85-90% if needed
* '''More settings:''' Click for advanced options
* '''Paper size:''' A4 (default) or Letter
* '''Margins:''' Default or narrow for more space

=== 🎯 Optimal Print Settings for Lab Protocols ===
{| class="wikitable"
!Setting
!Recommendation
!Why
|-
|'''Orientation'''
|Portrait
|For standard protocols
|-
|'''Scale'''
|85-90%
|Prevents text cutoff
|-
|'''Margins'''
|Narrow
|More space for content
|-
|'''Background graphics'''
|Off
|Saves ink
|-
|'''Headers/Footers'''
|Optional
|Print URL and date
|}

=== 💡 Pro Tips ===
'''Before printing:'''

* '''Check print preview:''' Is everything fully visible?
* '''Consider page count:''' Split long protocols?
* '''Images/Tables:''' Are they displayed correctly?

'''For large tables:'''

# Choose '''landscape orientation''' for wide pipetting schemes
# '''Reduce scale to 70-80%'''
# '''Check page breaks''' in preview

'''Save ink/toner:'''

* '''Disable background graphics'''
* '''Use draft mode''' for internal protocols
* '''Black & white''' for color printers

== 🔧 Troubleshooting ==

== Text gets cut off ==

* '''Reduce scale''' (85-90%)
* '''Set margins to "narrow"'''
* '''Use landscape''' for wide content

=== Page too long ===

* '''Check page breaks''' in preview
* '''Use "Print selection"''' for specific sections
* '''Create multiple PDFs''' for different sections

=== Images/formulas missing ===

* '''Enable "Print background graphics"'''
* '''Refresh browser''' before printing
* '''Take screenshot''' as alternative

----💡 '''Tip:''' For important protocols, always save as PDF first, then print!

== 🫱🏻‍🫲🏾 - Collaboration is key! ==
Knowledge grows when shared. That's why we appreciate it when you contribute your knowledge here and help us continue developing it.
 

== Getting started ==

* Configuration settings list
* MediaWiki FAQ
* MediaWiki release mailing list
* Localise MediaWiki for your language
* Learn how to combat spam on your wiki

Template:Labor-Boxes

2025-06-16T12:13:32Z

ESO wikiadmin:

<div style="display: grid; grid-template-columns: 1fr 1fr; gap: 20px; margin: 20px 0;">


<div style="background: #e8f4fd; border: 1px solid #3366cc; border-radius: 4px; padding: 16px; box-shadow: 0 1px 3px rgba(0,0,0,0.1);">
<h3 style="margin-top: 0; color: #0645ad; border-bottom: 1px solid #3366cc; padding-bottom: 8px; margin-bottom: 12px;">🧪 Protocols & more</h3>
<div>Documentation of all laboratory experiments and test procedures.
* All protocols from the VTK script and beyond. Important - Some protocols may still be out of date. Please let us know if you recognise an error: [[Methods and Protocols]] 
* Recipes for basic buffers and solutions in the laboratory [[Rezepte]] 
Only together can this wiki be useful and effective. If you have a protocol for a method that is worth implementing here, please contact ESO.
</div>
</div>


<div style="background: #f0f8f0; border: 1px solid #5cb85c; border-radius: 4px; padding: 16px; box-shadow: 0 1px 3px rgba(0,0,0,0.1);">
<h3 style="margin-top: 0; color: #0645ad; border-bottom: 1px solid #5cb85c; padding-bottom: 8px; margin-bottom: 12px;">⚡ Lab Devices</h3>
<div>Operating instructions, tips, and maintenance information for our lab equipment.
*[[Bio-Rad ChemiDoc Touch]]: Device for detecting Western blots
*[[Bio-Rad GelDoc]]: Device for analysing agarose gels
*[[Thermo Scientific Varioskan Flash]]: Multifunctional microplate reader
*[[PeqLab Thermocycler]]:
*[[Eppendorf Thermocycler]]
*[[Photometer]]
*[[S1 Inkubator]]
</div>
</div>


<div style="background: #fff5f0; border: 1px solid #ff6600; border-radius: 4px; padding: 16px; box-shadow: 0 1px 3px rgba(0,0,0,0.1);">
<h3 style="margin-top: 0; color: #0645ad; border-bottom: 1px solid #ff6600; padding-bottom: 8px; margin-bottom: 12px;">📋 Security & Safe Behaviour</h3>
<div>Safety regulations, emergency plans and protective measures.
*[[All About Safety]]: Be sure to read our internal laboratory information for more safety in the laboratory.
*Basic principles and guidelines can be found in the so-called laboratory guideline – Official web content of the University of Konstanz ([https://www.uni-konstanz.de/agu/arbeitssicherheit/spezifische-arbeitsbereiche/labore/|LINK]) and official DGUV information on biology laboratories ([https://res.jedermann.de/data/downloads/DGUV_Information_213-086.pdf|LINK])
In case of emergency: I-Point phone no.: 2222
</div>
</div>


<div style="background: #f5f0ff; border: 1px solid #8b5cf6; border-radius: 4px; padding: 16px; box-shadow: 0 1px 3px rgba(0,0,0,0.1);">
<h3 style="margin-top: 0; color: #0645ad; border-bottom: 1px solid #8b5cf6; padding-bottom: 8px; margin-bottom: 12px;">💡- Supporting things for your time in the lab</h3>
<div>
*[[Laboratory Bookkeeping Guide]]: How to keep written lab notes up to Electronic Lab Notebooks (ELN).
*How to write a [[Paper in 9 Steps]]: A guide on how to approach publishing a great paper someday.
*[[Software]]: Free programmes that are useful or indispensable in the lab.
*[[Forms and official documents]]: Important documents and corporate design template.
</div>

</div>
</div>

</div>

Plasmid-Miniprep (Birnboim – Dooley protocol)

2025-06-16T12:12:27Z

ESO wikiadmin:

{| class="wikitable"
|+
!Materials
|-
|Buffer P1: Solve 6.055 g Tris Base and 3.722g EDTA x 2H2O in 800 ml H20.
Adjust pH to 8.0 with 1M HCl, fill up to 1L.
Per 1 ml buffer P1, add 100 µg RNase A. Store at 4°C.
|-
|Buffer P2: Solve 8.0 g NaOH in 950 ml H20. Add 50 ml 20% SDS. Store at RT.
|-
|Puffer P3: 294.45g KAc in 500 ml H2O. Adjust pH to 5.5 with glacial acetic acid (about 110 ml acid needed), fill up to 1L with H2O. Store at 4°C.
|-
|TE-Puffer 10 mM Tris/HCl pH 8.0; 1mM EDTA
|-
|Isopropanol
|-
|sterile dd H2O 70% Ethanol
|-
|For each clone Klon 2 x 1,5ml Eppendorf cups
|}

== Procedure ==

* Resuspend completely bacterial pellet in 300µl P1 solution
* Add 300 µl P2 and invert tube 10x
* Add 300 µl P3 solution and invert tube 10x
* Centrifuge 10 min at 4°C (12.000rpm)
* Transfer 800 µl supernatant in a new tube and add 0.7 Vol. (560 µl) Isopropanol, invert 3x
* Centrifuge 15 min at 4°C (12.000rpm)
* Remove carefully supernatant and wash pellet with 0.5 ml 70% Ethanol
* Centrifuge 5 min (14,000rpm), remove supernatant
* Centrifuge pellet for additional 2 min at 14,000rpm
* Dry the pellet at 37°C (~ 15 min)
* Solve the pellet in 40 µl TE Puffer/ddH2O for at least 5 min
* Analyse quantity of plasmid DNA preparation

Western Blot: semi-dry blot

2025-06-16T12:11:13Z

ESO wikiadmin:

{| class="wikitable"
|+
!Materials
!
|-
|5X Anode buffer
|15 g Tris Base, pH to 10.4, final volume 1l
|-
|5X Catode buffer
|15 g Tris Base, 26.25 g 6-amino-N-caproic acid (6-Amino-hexansäure), pH 9.4; final volume 1 l
|}

=== Procedure ===
----

* Anode Buffer preparation: 20 ml 5x Anode buffer, 20 ml MeOH, fill up to 100 ml with ddH2O
* Cathode Buffer preparation: 20 ml 5x Cathode buffer, 20 ml MeOH, fill up to 100 ml with ddH2O
* The PVDF membrane is labeled with a pen and quickly activated in methanol, then rinsed with water and finally soaked in Anode Buffer
* Assemble the semi-dry transfer chamber and build the “transfer sandwich”: Soak 3x Whatman filter paper in Anode buffer and place on anode place the membrane on the stack of soaked Whatman paper place the SDS-PAGE gel on top of the membrane
* Soak 3x Whatman filter paper in Cathode buffer and place on the gel.
* Important: remove air bubbles trapped between the sandwich layers and especially between the membrane and the SDS-gel by using a roller
* Put the Kathode on top of the assembly and plug in the transfer chamber into the power supply
* Run the transfer at 50 mA (as a rule of thumb use 1 mA per cm² of the gel/membrane) for ~1:30h (for smaller proteins) or ~2h (for larger proteins); the time as well as the mA can be optimized for your protein of interest
* Upon completion of transfer, the apparatus is disassembled and the membrane is further processed as described in “Western Blot: probing of the membrane”

Lab Etiquette (ENG)

2025-06-16T09:19:50Z

ESO wikiadmin: /* Lab Book */

'''Diese Seite ist auch auf [[Lab Etiquette|Deutsch]] 🇩🇪 verfügbar.'''<div style="border: 1px solid #ccc; background: #f9f9f9; padding: 1em; margin-bottom: 1em;">
'''Welcome to the lab – think together, act together!'''

A well-functioning lab doesn’t rely solely on rules but on mutual awareness and shared responsibility. Especially for newcomers, it’s better to ask one question too many than to do the wrong thing silently. If you notice that something is running out – let people know. If you change something, inform others. That’s how we keep things smooth, safe, and effective.
</div>

= Laboratory Manual - Cell Biology Research Group =

== Laboratory Access ==
The ML laboratory building is open on weekdays from 6:30 AM to 8:30 PM. Access to laboratory rooms is obtained using RFID transponders, which also allow entry to the laboratory building outside the above opening hours. Master's students and doctoral candidates receive a personal transponder through key distribution (form available from Petra Schnurr) and must return the transponder after completing their time as employees. For bachelor's students and research assistants, a loan transponder is available in exceptional cases after training for necessary work outside opening hours. The loan transponder is located in the key cabinet at the general computer workstation. Removal of the loan transponder must be noted in the list with date and initials.

== Working Hours ==
Non-scientific staff and research assistants are required to record their working time. The online website of the personnel administration is available for this purpose:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

Research assistants use timesheets for time recording (available from Petra Schnurr).

Scientific staff should be present during core working hours 9:00 AM – 12:00 PM and 2:00 PM – 4:00 PM. Absence must be discussed with Christof and noted in the wall calendar in the entrance area of the office row.

== Illness ==
In case of illness, Christof and the secretariat should be notified as soon as possible by telephone and/or email so that coverage, etc. can be organized. For prolonged illness, a medical certificate/medical attestation must be sent to petra.schnurr@uni-konstanz.de on the third day of absence.

== Vacation ==
Employees should apply for vacation as early as possible (at least 1 week in advance) through the online time recording system:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

The possibility for long-term vacation (> 2 weeks) is project-dependent and should be discussed with Christof at least 2 months in advance. During the second half of the winter semester (early January – end of February), vacation during the VTK is only possible in exceptional cases.

Before taking vacation, the culture/storage of important clones/cell lines/samples must always be ensured and coverage for lab duties must be organized. Bachelor's/Master's students should discuss multi-day absences with their supervisor and Christof.

== Seminars/Project Meetings ==
Monday mornings (8:45 AM) there is a general lab meeting. This is followed by project presentations by individual staff members, which are announced on the notice board in the entrance area.

Wednesday afternoons from 3:15 PM – approximately 5:00 PM our journal club takes place. The publication being discussed is loaded onto the file server or sent as a PDF file to everyone the week before. Every two weeks in this seminar, we discuss current new data from all staff members.

Participation in both seminars is mandatory; the seminar schedule is usually set three to six months in advance.

Tuesday afternoons and Thursday lunchtimes during the lecture period, the seminars of the graduate school KoRS-CB as well as the departmental seminar of the Biology Department take place. Speakers/topics are announced by posting. Participation is optional, but when possible, one should not miss interesting presentations.

Specific project meetings take place by arrangement with Christof. Lab books should be brought to project meetings as a basis for discussion.

== Name Abbreviations/Labels ==
Each employee uses a distinctive name abbreviation with which all samples, solutions, etc. must be labeled. Name abbreviations are assigned by the secretariat and are stored in a list on the file server:

Fileserver_LSH/Templates/Lab-Directories/Name-Abbreviations_AG Hauck_2025.doc

== Lab Book ==
Each employee maintains a lab book, which is issued by the secretariat and in which all work is documented in chronological order. Templates for documenting various experiments are stored as PDF files on the file server:

Fileserver_LSH/Templates/Lab-Documentation/

* Gentamicin Assay: "Steffi"
* PCR: "PCR-based-LIC-cloning-protocol-CRH.pdf"
* Cell transfection: "HEKcell-transfection-Protocol-JWK.pdf"
* In vitro Kinase Assay: "Erik"
* [[Immunoprecipitation]] (MHE)
* [[Protein expression and purification]] (SBA)
* Lentivirus production: "Marlene"
* Bacterial growth curve: "Ann-Kathrin"

The pages of the lab book are numbered consecutively and a table of contents is created on the first two pages.

The lab book belongs to the Cell Biology Department and remains the property of the department after departure. No pages may be removed from the lab book. Original printouts, gel copies, X-ray films, etc. are labeled and attached to the lab book by being stapled or glued to the corresponding experiments. Electronic original data is stored on the file server in a separate user directory (see Data Archiving chapter).

For most experiments, there are already lab protocols that are described in detail in our VTK script and can be referenced in the lab book. The VTK script can be found on the file server:

''Fileserver_LSH/Templates/Lab-Documentation/Script-Advanced-Course-2025.doc''

The reagents and solutions used are also described either in the recipe file: ''Fileserver_LSH/Recipes/Recipes-2025.doc'' or can be found in the chemical list:

''Fileserver_LSH/Chemical-List/Chemical-List 2025 Group Hauck.xls''

'''Only when deviating from existing protocols are special notes in the lab book necessary.'''

For microscopic work, flow cytometric analyses, or similar work with results in electronic form, a sample protocol is prepared (see Fileserver_LSH/Templates/Lab-Documentation/Protocol-Microscope-FACS.doc") and attached to the experiment.

During project meetings, the lab book serves as the basis for discussing results and should therefore definitely be brought along.

== Data Archiving ==
Standard experiments should be performed according to the lab protocols listed above; deviations should be recorded in the lab book. The raw data obtained during experiments (e.g., microscopy images, gel images, FACS data, VarioScan data) as well as derived figures created from them (graphics, processed microscopy images, etc.) should be stored on the data file server "Fileserver_LSH_Data/00_User/" in a separate user folder. Only there is automatic (daily) backup performed, so no data can be lost.

Each electronic data file contains the date, name abbreviation, and experiment in the name (The raw data files are named according to the following scheme):

'''Year-Month-Day_Name-Abbreviation_Experiment / year-month-day_initials_experiment'''

e.g. /e.g.: 2025-01-01_CRH_Adhesion assay

* the digital version/raw files should be on the file server with the date that can also be found in the lab book
* a printed version of the evaluated data should be attached in the lab book

For later use in publications it is important that we all stick to the same folder structure and naming of files. An example of such a user folder is given on the Fileserver and depicted on the next page:

Fileserver_LSH_Data/00_User/xUE_Universal_Example/

'''Data'''

* ''Constructs''
** SuperSH2 agarose gels, sequencing results, plasmid map, etc.
* ''Western Blots''
* ''Immunofluorescence staining''
* ''Flow Cytometry''
* ''ELISA/SEAP-Assays''
* ''...''

'''Literature'''

'''Presentations'''

'''Thesis:''' for your thesis: for '''every''' figure in your thesis create a subfolder named with the Figure-Number and include in each subfolder both: the final figure as pdf- and illustrator-file and the '''used raw data''' (subdivided in panels A, B, etc.). The name of the raw data file should remain the same as it is found in your DATA-folder and lab book)
[[File:Labetiquette fileserver.jpg|thumb|center|696x696px|'''Fig. 1:''' Example of the user folder structure on the Fileserver_LSH_Data of user UE_Universal_Example.]]

== Laboratory Workspace ==
In the laboratories, there are general laboratory workspaces (e.g., in the two cell culture labs, in the infection room, or the area with protein gel electrophoresis) as well as private workspaces that are assigned. Work at private workspaces of others should only occur after consultation. Everyone is responsible for keeping their workspace, associated shelves, and drawers clean. The work surface is wiped down and disinfected at least once per week; drawers/shelves should be dusted regularly and kept tidy.

Additionally, everyone has a storage space in a refrigerator at 4°C, in a freezer/compartment at -20°C, and can store samples in labeled boxes in the -80°C freezer in room ML. Plastic boxes with lids are available for storage; open stands (especially for Eppendorf tubes) should only be used for short-term storage (1 day). All samples (Eppendorf tubes, agar plates, etc.) must be labeled with abbreviation, content description, and date. All storage locations should be organized/updated regularly (at least once per month). For storage in the -80°C freezer, a data sheet must be filed in the -80°C folder for each box. A template for the data sheet can be found on the file server:

''Fileserver_LSH/Templates/Lab-Documentation/-80C Box-Template.doc''

Please reduce opening of deep freezers to a minimum (otherwise icing and malfunction). Therefore, think before opening about what should be removed and where it can be found (see data sheet!!!). General laboratory workspaces should be cleaned after each use (e.g., sterile bench, protein gel electrophoresis, fume hood, scales, gel documentation) and all used solutions, equipment, etc. must be put away.

== Lab Jobs ==
Each employee receives a lab job that they perform for the general community. The lab job can include maintaining a general collection, responsibility for a device, or supervision and organization of a laboratory. In addition, the preparation of solutions/buffers etc. for the general community is handled through lab jobs. Those responsible are listed on the notice board at the entrance. Room supervisors also organize regular maintenance/cleaning schedules for general equipment such as sterile benches, incubators, etc.

== Closing Service ==
The evening closing service, which ensures that equipment such as sterile benches, vacuum pumps, gel documentation, or the propane gas line are turned off, rotates weekly among scientific staff and master's students. A list for the evening control round of the closing service hangs on the notice board at the entrance.

== Computer Use ==
There is a computer for general use in the seminar room. Please always enable use for official purposes. Private PCs/notebooks can be connected to the network. A current virus scanner and activation of a firewall are required. Access to the department's network storage in the computing center (Fileserver_LSH and Fileserver_LSH_Data) is set up by Christof. Various software programs are available on Fileserver_LSH_Data/02_SOFTWARE, including Adobe Photoshop CS4, Adobe Acrobat Pro9, Adobe Illustrator CS4, MS Office 2016, Clone Manager 9, Endnote X9, SigmaStat 4.0, Leica LAS, Biorad QuantityOne, VarioScan SkanIt. To ensure smooth data exchange within the lab, the above-mentioned versions of these programs must be used for all official purposes, or the data must be saved in a format compatible with these program versions.

== Everyone's Cooperation is Required ==
Each employee can contribute with little effort to minimizing additional work in the laboratory for everyone. Thus, everyone should keep consumption/waste/cleanup effort as low as possible through forward-thinking planning and also help with waste disposal. Please do not fill autoclave bags until "overflowing," but close them in time, deposit them in the tray, and put a new autoclave bag in the corresponding waste container.

Everyone is also responsible for filling boxes with pipette tips and refilling Eppendorf tubes. Autoclave materials are collected in the plastic box and autoclaved by Claudia/Susanne for everyone.

Proper behavior also includes conduct and cleanliness in the tea kitchen and social room. If I have never loaded or unloaded the dishwasher or refilled the coffee machine during the course of one month, then I have very likely used clean dishes at the expense of others.

== Chemicals/Solutions ==
Many solutions are prepared as lab jobs and either kept accessible to everyone as stock solutions in designated places or provided as aliquoted solutions, where one takes an aliquot to their own workspace as needed and stores it there for further use. Additional solutions for personal use should be prepared yourself (please estimate the required amounts in advance and do not prepare liter-wise solutions when you only need 10 ml for two experiments in total) and correctly labeled (abbreviation, date, content: designation and concentration; hazard symbol) and stored at your own workspace. Use solutions and aliquots at colleagues' workspaces ONLY after consultation. Stock solutions, aliquoted solutions, and the respective responsible persons are listed on the "Lab Jobs" bulletin board. For preparing solutions, there is a recipe book available as a file on the file server:

Fileserver_LSH/Recipes/Recipes-2025.doc

== General Collections ==
The department maintains various collections that represent an important resource for everyone and must therefore be handled with particular care and responsibility.

'''Strain Collection:''' includes all bacterial strains that are filed by numbers. Storage at -70°C in chest on level 5 as well as in room ML6:

* non-pathogenic bacteria (= simultaneously repository of all produced plasmids)
* pathogenic bacteria and mutants (key available from Christof)

The strain collection is also available as an MS Access file. Each newly produced or externally obtained bacterial strain receives a new strain number and is frozen in the strain collection as well as electronically recorded. Number distribution is handled by Christof. Only Susanne and Christof have access to the strain collection.

'''Plasmid Collection:''' includes all isolated plasmids from non-pathogenic bacterial strains of the strain collection. Numbering is identical to the strain collection (storage in general refrigerator at 4°C)

'''Cell Collection:''' includes stocks of all cell lines and primary cells. Stored in liquid nitrogen in the media kitchen. The cell collection is managed by Susanne.

'''Virus Collection:''' includes all lentiviral, adenoviral, AAV particles, and bacteriophages. Stored at -70°C. Access file!

'''Oligo Collection:''' includes all oligos ordered for cloning, PCRs, etc. Oligos are stored as 10 mM stock solution in TE buffer at -20°C in the general collection, not in private freezers. Ordering new oligos and filing information is handled by Jan Kuiper.

'''Antibody Collection:''' Collection of all primary antibodies (commercial or self-made) and secondary antibodies (enzyme- or fluorescence-conjugated). Data sheets are collected in the "Antibody" folder. Claudia Hentschel organizes the antibody collection.

'''Protein Collection:''' All recombinant, purified proteins are recorded here.

'''Inhibitor Collection:''' Information on all pharmacological inhibitors and the concentration, solvent, and storage location of stock solutions are filed here. _______________ organizes the inhibitor collection.

'''Enzyme Collection:''' includes all commercial and self-made (DpnI) restriction enzymes stored at -20°C. Removal of enzymes from stock containers only with FILTER TIPS!

Enzymes may only be removed from the freezer for the shortest possible time (1-2 minutes) (i.e., directly before use) and are stored during this short time in a paraffin block cooled to -20°C.

''Folders with documentation for all collections can be found on the shelves at the general workspace in the office area.''

== Competent Cells ==
Aliquots of competent bacteria are stored at -80°C. 200 μl are frozen per aliquot, sufficient for 2 transformations. Various strains are available for different purposes. The standard cloning strain is NovaBlue. When removing, proceed as quickly as possible; warming of aliquots leads to reduced competence; do not remove more aliquots than actually needed.

== Enzymes ==
Polymerases, restriction enzymes, etc. are stored at -20°C in boxes or on paraffin blocks together with the corresponding buffers. These enzymes are used jointly by everyone. Therefore, handle enzyme stock solutions particularly cleanly and avoid contamination (filter tips!!!) as well as warming.

When removing enzymes, transport the Eppendorf tubes with the stock solution exclusively in a paraffin block cooled to -20°C to the workplace and leave them in it for pipetting. Even with paraffin block, remove the enzyme from the -20°C freezer only briefly (< 2 minutes) and return it immediately after pipetting. When opening stock containers, definitely avoid contact of the inner lid surface with skin!!

== Orders ==
When general chemicals or disposables are running low, give notice IN TIME (i.e., before using up the last package or pipetting the last microliter): either write on the board in the lab or tell the orderers. Discuss project-specific chemicals/antibodies etc. or lab utensils/equipment with Christof before ordering.

For oligo orders through Jan, enter the oligo information in the order list on the file server: Fileserver_LSH:\Strain-Collection\Primer\01_PrimerOrdering_Primer-Order

Discuss other online orders with Christof.

Order sequencing using pre-paid labels from LGC or GeneWiz. Please no more than 2 sequencing per new construct. Discuss larger sequencing needs with Christof. Deliveries of reagents should be handed over to the orderer. The orderer checks for completeness (using the delivery slip) and confirms with date and signature. The delivery slip and any accompanying invoices are forwarded to Petra Schnurr in the secretariat. When chemicals, antibodies, enzymes, and other reagents are delivered, the data sheets are dated and filed in the corresponding folders (see "General Collections"). The solvent, concentration, and possibly filled aliquots as well as storage location may be noted on the data sheets.

ALL chemicals, reagents, enzymes, etc. are stored in accessible and known locations.

== Laboratory Safety ==
See [[All About Safety|Safety Guidelines]].

Lab Etiquette (ENG)

2025-06-16T09:19:25Z

ESO wikiadmin:

'''Diese Seite ist auch auf [[Lab Etiquette|Deutsch]] 🇩🇪 verfügbar.'''<div style="border: 1px solid #ccc; background: #f9f9f9; padding: 1em; margin-bottom: 1em;">
'''Welcome to the lab – think together, act together!'''

A well-functioning lab doesn’t rely solely on rules but on mutual awareness and shared responsibility. Especially for newcomers, it’s better to ask one question too many than to do the wrong thing silently. If you notice that something is running out – let people know. If you change something, inform others. That’s how we keep things smooth, safe, and effective.
</div>

= Laboratory Manual - Cell Biology Research Group =

== Laboratory Access ==
The ML laboratory building is open on weekdays from 6:30 AM to 8:30 PM. Access to laboratory rooms is obtained using RFID transponders, which also allow entry to the laboratory building outside the above opening hours. Master's students and doctoral candidates receive a personal transponder through key distribution (form available from Petra Schnurr) and must return the transponder after completing their time as employees. For bachelor's students and research assistants, a loan transponder is available in exceptional cases after training for necessary work outside opening hours. The loan transponder is located in the key cabinet at the general computer workstation. Removal of the loan transponder must be noted in the list with date and initials.

== Working Hours ==
Non-scientific staff and research assistants are required to record their working time. The online website of the personnel administration is available for this purpose:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

Research assistants use timesheets for time recording (available from Petra Schnurr).

Scientific staff should be present during core working hours 9:00 AM – 12:00 PM and 2:00 PM – 4:00 PM. Absence must be discussed with Christof and noted in the wall calendar in the entrance area of the office row.

== Illness ==
In case of illness, Christof and the secretariat should be notified as soon as possible by telephone and/or email so that coverage, etc. can be organized. For prolonged illness, a medical certificate/medical attestation must be sent to petra.schnurr@uni-konstanz.de on the third day of absence.

== Vacation ==
Employees should apply for vacation as early as possible (at least 1 week in advance) through the online time recording system:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

The possibility for long-term vacation (> 2 weeks) is project-dependent and should be discussed with Christof at least 2 months in advance. During the second half of the winter semester (early January – end of February), vacation during the VTK is only possible in exceptional cases.

Before taking vacation, the culture/storage of important clones/cell lines/samples must always be ensured and coverage for lab duties must be organized. Bachelor's/Master's students should discuss multi-day absences with their supervisor and Christof.

== Seminars/Project Meetings ==
Monday mornings (8:45 AM) there is a general lab meeting. This is followed by project presentations by individual staff members, which are announced on the notice board in the entrance area.

Wednesday afternoons from 3:15 PM – approximately 5:00 PM our journal club takes place. The publication being discussed is loaded onto the file server or sent as a PDF file to everyone the week before. Every two weeks in this seminar, we discuss current new data from all staff members.

Participation in both seminars is mandatory; the seminar schedule is usually set three to six months in advance.

Tuesday afternoons and Thursday lunchtimes during the lecture period, the seminars of the graduate school KoRS-CB as well as the departmental seminar of the Biology Department take place. Speakers/topics are announced by posting. Participation is optional, but when possible, one should not miss interesting presentations.

Specific project meetings take place by arrangement with Christof. Lab books should be brought to project meetings as a basis for discussion.

== Name Abbreviations/Labels ==
Each employee uses a distinctive name abbreviation with which all samples, solutions, etc. must be labeled. Name abbreviations are assigned by the secretariat and are stored in a list on the file server:

Fileserver_LSH/Templates/Lab-Directories/Name-Abbreviations_AG Hauck_2025.doc

== Lab Book ==
Each employee maintains a lab book, which is issued by the secretariat and in which all work is documented in chronological order. Templates for documenting various experiments are stored as PDF files on the file server:

Fileserver_LSH/Templates/Lab-Documentation/

* Gentamicin Assay: "Steffi"
* PCR: "PCR-based-LIC-cloning-protocol-CRH.pdf"
* Cell transfection: "HEKcell-transfection-Protocol-JWK.pdf"
* In vitro Kinase Assay: "Erik"
* [[Immunoprecipitation]] (MHE)
* Protein expression and purification: "Sarah"
* Lentivirus production: "Marlene"
* Bacterial growth curve: "Ann-Kathrin"

The pages of the lab book are numbered consecutively and a table of contents is created on the first two pages.

The lab book belongs to the Cell Biology Department and remains the property of the department after departure. No pages may be removed from the lab book. Original printouts, gel copies, X-ray films, etc. are labeled and attached to the lab book by being stapled or glued to the corresponding experiments. Electronic original data is stored on the file server in a separate user directory (see Data Archiving chapter).

For most experiments, there are already lab protocols that are described in detail in our VTK script and can be referenced in the lab book. The VTK script can be found on the file server:

''Fileserver_LSH/Templates/Lab-Documentation/Script-Advanced-Course-2025.doc''

The reagents and solutions used are also described either in the recipe file: ''Fileserver_LSH/Recipes/Recipes-2025.doc'' or can be found in the chemical list:

''Fileserver_LSH/Chemical-List/Chemical-List 2025 Group Hauck.xls''

'''Only when deviating from existing protocols are special notes in the lab book necessary.'''

For microscopic work, flow cytometric analyses, or similar work with results in electronic form, a sample protocol is prepared (see Fileserver_LSH/Templates/Lab-Documentation/Protocol-Microscope-FACS.doc") and attached to the experiment.

During project meetings, the lab book serves as the basis for discussing results and should therefore definitely be brought along.

== Data Archiving ==
Standard experiments should be performed according to the lab protocols listed above; deviations should be recorded in the lab book. The raw data obtained during experiments (e.g., microscopy images, gel images, FACS data, VarioScan data) as well as derived figures created from them (graphics, processed microscopy images, etc.) should be stored on the data file server "Fileserver_LSH_Data/00_User/" in a separate user folder. Only there is automatic (daily) backup performed, so no data can be lost.

Each electronic data file contains the date, name abbreviation, and experiment in the name (The raw data files are named according to the following scheme):

'''Year-Month-Day_Name-Abbreviation_Experiment / year-month-day_initials_experiment'''

e.g. /e.g.: 2025-01-01_CRH_Adhesion assay

* the digital version/raw files should be on the file server with the date that can also be found in the lab book
* a printed version of the evaluated data should be attached in the lab book

For later use in publications it is important that we all stick to the same folder structure and naming of files. An example of such a user folder is given on the Fileserver and depicted on the next page:

Fileserver_LSH_Data/00_User/xUE_Universal_Example/

'''Data'''

* ''Constructs''
** SuperSH2 agarose gels, sequencing results, plasmid map, etc.
* ''Western Blots''
* ''Immunofluorescence staining''
* ''Flow Cytometry''
* ''ELISA/SEAP-Assays''
* ''...''

'''Literature'''

'''Presentations'''

'''Thesis:''' for your thesis: for '''every''' figure in your thesis create a subfolder named with the Figure-Number and include in each subfolder both: the final figure as pdf- and illustrator-file and the '''used raw data''' (subdivided in panels A, B, etc.). The name of the raw data file should remain the same as it is found in your DATA-folder and lab book)
[[File:Labetiquette fileserver.jpg|thumb|center|696x696px|'''Fig. 1:''' Example of the user folder structure on the Fileserver_LSH_Data of user UE_Universal_Example.]]

== Laboratory Workspace ==
In the laboratories, there are general laboratory workspaces (e.g., in the two cell culture labs, in the infection room, or the area with protein gel electrophoresis) as well as private workspaces that are assigned. Work at private workspaces of others should only occur after consultation. Everyone is responsible for keeping their workspace, associated shelves, and drawers clean. The work surface is wiped down and disinfected at least once per week; drawers/shelves should be dusted regularly and kept tidy.

Additionally, everyone has a storage space in a refrigerator at 4°C, in a freezer/compartment at -20°C, and can store samples in labeled boxes in the -80°C freezer in room ML. Plastic boxes with lids are available for storage; open stands (especially for Eppendorf tubes) should only be used for short-term storage (1 day). All samples (Eppendorf tubes, agar plates, etc.) must be labeled with abbreviation, content description, and date. All storage locations should be organized/updated regularly (at least once per month). For storage in the -80°C freezer, a data sheet must be filed in the -80°C folder for each box. A template for the data sheet can be found on the file server:

''Fileserver_LSH/Templates/Lab-Documentation/-80C Box-Template.doc''

Please reduce opening of deep freezers to a minimum (otherwise icing and malfunction). Therefore, think before opening about what should be removed and where it can be found (see data sheet!!!). General laboratory workspaces should be cleaned after each use (e.g., sterile bench, protein gel electrophoresis, fume hood, scales, gel documentation) and all used solutions, equipment, etc. must be put away.

== Lab Jobs ==
Each employee receives a lab job that they perform for the general community. The lab job can include maintaining a general collection, responsibility for a device, or supervision and organization of a laboratory. In addition, the preparation of solutions/buffers etc. for the general community is handled through lab jobs. Those responsible are listed on the notice board at the entrance. Room supervisors also organize regular maintenance/cleaning schedules for general equipment such as sterile benches, incubators, etc.

== Closing Service ==
The evening closing service, which ensures that equipment such as sterile benches, vacuum pumps, gel documentation, or the propane gas line are turned off, rotates weekly among scientific staff and master's students. A list for the evening control round of the closing service hangs on the notice board at the entrance.

== Computer Use ==
There is a computer for general use in the seminar room. Please always enable use for official purposes. Private PCs/notebooks can be connected to the network. A current virus scanner and activation of a firewall are required. Access to the department's network storage in the computing center (Fileserver_LSH and Fileserver_LSH_Data) is set up by Christof. Various software programs are available on Fileserver_LSH_Data/02_SOFTWARE, including Adobe Photoshop CS4, Adobe Acrobat Pro9, Adobe Illustrator CS4, MS Office 2016, Clone Manager 9, Endnote X9, SigmaStat 4.0, Leica LAS, Biorad QuantityOne, VarioScan SkanIt. To ensure smooth data exchange within the lab, the above-mentioned versions of these programs must be used for all official purposes, or the data must be saved in a format compatible with these program versions.

== Everyone's Cooperation is Required ==
Each employee can contribute with little effort to minimizing additional work in the laboratory for everyone. Thus, everyone should keep consumption/waste/cleanup effort as low as possible through forward-thinking planning and also help with waste disposal. Please do not fill autoclave bags until "overflowing," but close them in time, deposit them in the tray, and put a new autoclave bag in the corresponding waste container.

Everyone is also responsible for filling boxes with pipette tips and refilling Eppendorf tubes. Autoclave materials are collected in the plastic box and autoclaved by Claudia/Susanne for everyone.

Proper behavior also includes conduct and cleanliness in the tea kitchen and social room. If I have never loaded or unloaded the dishwasher or refilled the coffee machine during the course of one month, then I have very likely used clean dishes at the expense of others.

== Chemicals/Solutions ==
Many solutions are prepared as lab jobs and either kept accessible to everyone as stock solutions in designated places or provided as aliquoted solutions, where one takes an aliquot to their own workspace as needed and stores it there for further use. Additional solutions for personal use should be prepared yourself (please estimate the required amounts in advance and do not prepare liter-wise solutions when you only need 10 ml for two experiments in total) and correctly labeled (abbreviation, date, content: designation and concentration; hazard symbol) and stored at your own workspace. Use solutions and aliquots at colleagues' workspaces ONLY after consultation. Stock solutions, aliquoted solutions, and the respective responsible persons are listed on the "Lab Jobs" bulletin board. For preparing solutions, there is a recipe book available as a file on the file server:

Fileserver_LSH/Recipes/Recipes-2025.doc

== General Collections ==
The department maintains various collections that represent an important resource for everyone and must therefore be handled with particular care and responsibility.

'''Strain Collection:''' includes all bacterial strains that are filed by numbers. Storage at -70°C in chest on level 5 as well as in room ML6:

* non-pathogenic bacteria (= simultaneously repository of all produced plasmids)
* pathogenic bacteria and mutants (key available from Christof)

The strain collection is also available as an MS Access file. Each newly produced or externally obtained bacterial strain receives a new strain number and is frozen in the strain collection as well as electronically recorded. Number distribution is handled by Christof. Only Susanne and Christof have access to the strain collection.

'''Plasmid Collection:''' includes all isolated plasmids from non-pathogenic bacterial strains of the strain collection. Numbering is identical to the strain collection (storage in general refrigerator at 4°C)

'''Cell Collection:''' includes stocks of all cell lines and primary cells. Stored in liquid nitrogen in the media kitchen. The cell collection is managed by Susanne.

'''Virus Collection:''' includes all lentiviral, adenoviral, AAV particles, and bacteriophages. Stored at -70°C. Access file!

'''Oligo Collection:''' includes all oligos ordered for cloning, PCRs, etc. Oligos are stored as 10 mM stock solution in TE buffer at -20°C in the general collection, not in private freezers. Ordering new oligos and filing information is handled by Jan Kuiper.

'''Antibody Collection:''' Collection of all primary antibodies (commercial or self-made) and secondary antibodies (enzyme- or fluorescence-conjugated). Data sheets are collected in the "Antibody" folder. Claudia Hentschel organizes the antibody collection.

'''Protein Collection:''' All recombinant, purified proteins are recorded here.

'''Inhibitor Collection:''' Information on all pharmacological inhibitors and the concentration, solvent, and storage location of stock solutions are filed here. _______________ organizes the inhibitor collection.

'''Enzyme Collection:''' includes all commercial and self-made (DpnI) restriction enzymes stored at -20°C. Removal of enzymes from stock containers only with FILTER TIPS!

Enzymes may only be removed from the freezer for the shortest possible time (1-2 minutes) (i.e., directly before use) and are stored during this short time in a paraffin block cooled to -20°C.

''Folders with documentation for all collections can be found on the shelves at the general workspace in the office area.''

== Competent Cells ==
Aliquots of competent bacteria are stored at -80°C. 200 μl are frozen per aliquot, sufficient for 2 transformations. Various strains are available for different purposes. The standard cloning strain is NovaBlue. When removing, proceed as quickly as possible; warming of aliquots leads to reduced competence; do not remove more aliquots than actually needed.

== Enzymes ==
Polymerases, restriction enzymes, etc. are stored at -20°C in boxes or on paraffin blocks together with the corresponding buffers. These enzymes are used jointly by everyone. Therefore, handle enzyme stock solutions particularly cleanly and avoid contamination (filter tips!!!) as well as warming.

When removing enzymes, transport the Eppendorf tubes with the stock solution exclusively in a paraffin block cooled to -20°C to the workplace and leave them in it for pipetting. Even with paraffin block, remove the enzyme from the -20°C freezer only briefly (< 2 minutes) and return it immediately after pipetting. When opening stock containers, definitely avoid contact of the inner lid surface with skin!!

== Orders ==
When general chemicals or disposables are running low, give notice IN TIME (i.e., before using up the last package or pipetting the last microliter): either write on the board in the lab or tell the orderers. Discuss project-specific chemicals/antibodies etc. or lab utensils/equipment with Christof before ordering.

For oligo orders through Jan, enter the oligo information in the order list on the file server: Fileserver_LSH:\Strain-Collection\Primer\01_PrimerOrdering_Primer-Order

Discuss other online orders with Christof.

Order sequencing using pre-paid labels from LGC or GeneWiz. Please no more than 2 sequencing per new construct. Discuss larger sequencing needs with Christof. Deliveries of reagents should be handed over to the orderer. The orderer checks for completeness (using the delivery slip) and confirms with date and signature. The delivery slip and any accompanying invoices are forwarded to Petra Schnurr in the secretariat. When chemicals, antibodies, enzymes, and other reagents are delivered, the data sheets are dated and filed in the corresponding folders (see "General Collections"). The solvent, concentration, and possibly filled aliquots as well as storage location may be noted on the data sheets.

ALL chemicals, reagents, enzymes, etc. are stored in accessible and known locations.

== Laboratory Safety ==
See [[All About Safety|Safety Guidelines]].

Protein expression and purification

2025-06-05T13:43:44Z

ESO wikiadmin:

''This is an original excerpt from SBAs digital labbook from May 2025.''

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
[[File:SB20.png|right|frameless|328x328px]]
* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==

* Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
[[File:SBA 21.png|right|frameless|

*

]]

* Dialysis performed by SF
* Mix samples with 2x SDS loading buffer

* Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

* Perform SDS-Page at 80V/120V

* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
[[File:SBA 24.png|right|frameless|389x389px]]
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)
* The column is hung upside down
* Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)
* Remove the tube from the end of the column and insert column the right way round
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL
* Prepare 6 Schnappdeckelröhrchen
* Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)
* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here
* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1
* Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2
* Wash column with elution buffer 2 for 15 min at 0.5 mL/min
* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page
* Run SDS-Page at 80V /120 V
* Stain gel with coomassie for 45 min at rt after short warming in microwave
* Destain until bands are visible

''Cleaning of Column after use:''

* wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)
* wash 20 min 1 mL/min with Wash buffer (20 mL)
* wash 20 mL 1 mL/min with H2O (degassed)
* wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
[[File:SBA 25.png|right|frameless|243x243px]]
* Mix samples with 2x SDS loading buffer
* Boil samples 95°C for 5 min
* Load onto a 12.5% acrylamide gel
* Run at 80 V/120 V
* Coomassie stain after short heating in microwave for 40 min
* Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==

* Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)
** Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate
** Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)
** Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.
** Measure absorbance at 660 nm
** Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer
[[File:SBA curve.png|center|frameless|604x604px]]

Protein expression and purification

2025-06-05T13:42:42Z

ESO wikiadmin: /* Concentration Determination of purified recombinant hPOPX2 (PPM1F) */

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
[[File:SB20.png|right|frameless|328x328px]]
* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==

* Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
[[File:SBA 21.png|right|frameless|

*

]]

* Dialysis performed by SF
* Mix samples with 2x SDS loading buffer

* Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

* Perform SDS-Page at 80V/120V

* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
[[File:SBA 24.png|right|frameless|389x389px]]
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)
* The column is hung upside down
* Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)
* Remove the tube from the end of the column and insert column the right way round
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL
* Prepare 6 Schnappdeckelröhrchen
* Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)
* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here
* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1
* Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2
* Wash column with elution buffer 2 for 15 min at 0.5 mL/min
* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page
* Run SDS-Page at 80V /120 V
* Stain gel with coomassie for 45 min at rt after short warming in microwave
* Destain until bands are visible

''Cleaning of Column after use:''

* wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)
* wash 20 min 1 mL/min with Wash buffer (20 mL)
* wash 20 mL 1 mL/min with H2O (degassed)
* wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
[[File:SBA 25.png|right|frameless|243x243px]]
* Mix samples with 2x SDS loading buffer
* Boil samples 95°C for 5 min
* Load onto a 12.5% acrylamide gel
* Run at 80 V/120 V
* Coomassie stain after short heating in microwave for 40 min
* Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==

* Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)
** Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate
** Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)
** Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.
** Measure absorbance at 660 nm
** Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer
[[File:SBA curve.png|center|frameless|604x604px]]

File:SBA curve.png

2025-06-05T13:42:24Z

ESO wikiadmin:

SBA curve

Protein expression and purification

2025-06-05T13:41:04Z

ESO wikiadmin: /* Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26 */

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
[[File:SB20.png|right|frameless|328x328px]]
* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==

* Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
[[File:SBA 21.png|right|frameless|

*

]]

* Dialysis performed by SF
* Mix samples with 2x SDS loading buffer

* Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

* Perform SDS-Page at 80V/120V

* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
[[File:SBA 24.png|right|frameless|389x389px]]
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)
* The column is hung upside down
* Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)
* Remove the tube from the end of the column and insert column the right way round
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL
* Prepare 6 Schnappdeckelröhrchen
* Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)
* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here
* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1
* Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2
* Wash column with elution buffer 2 for 15 min at 0.5 mL/min
* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page
* Run SDS-Page at 80V /120 V
* Stain gel with coomassie for 45 min at rt after short warming in microwave
* Destain until bands are visible

''Cleaning of Column after use:''

* wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)
* wash 20 min 1 mL/min with Wash buffer (20 mL)
* wash 20 mL 1 mL/min with H2O (degassed)
* wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
[[File:SBA 25.png|right|frameless|243x243px]]
* Mix samples with 2x SDS loading buffer
* Boil samples 95°C for 5 min
* Load onto a 12.5% acrylamide gel
* Run at 80 V/120 V
* Coomassie stain after short heating in microwave for 40 min
* Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)

o Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate

o Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)

o Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.

o Measure absorbance at 660 nm

o Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer

File:SBA 25.png

2025-06-05T13:40:43Z

ESO wikiadmin:

SBA 25

Protein expression and purification

2025-06-05T13:39:36Z

ESO wikiadmin: /* Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2 */

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
[[File:SB20.png|right|frameless|328x328px]]
* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==

* Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
[[File:SBA 21.png|right|frameless|

*

]]

* Dialysis performed by SF
* Mix samples with 2x SDS loading buffer

* Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

* Perform SDS-Page at 80V/120V

* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
[[File:SBA 24.png|right|frameless|389x389px]]
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)
* The column is hung upside down
* Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)
* Remove the tube from the end of the column and insert column the right way round
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL
* Prepare 6 Schnappdeckelröhrchen
* Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)
* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here
* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1
* Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2
* Wash column with elution buffer 2 for 15 min at 0.5 mL/min
* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page
* Run SDS-Page at 80V /120 V
* Stain gel with coomassie for 45 min at rt after short warming in microwave
* Destain until bands are visible

''Cleaning of Column after use:''

* wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)
* wash 20 min 1 mL/min with Wash buffer (20 mL)
* wash 20 mL 1 mL/min with H2O (degassed)
* wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==

* Mix samples with 2x SDS loading buffer
* Boil samples 95°C for 5 min
* Load onto a 12.5% acrylamide gel
* Run at 80 V/120 V
* Coomassie stain after short heating in microwave for 40 min
* Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)

o Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate

o Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)

o Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.

o Measure absorbance at 660 nm

o Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer

Protein expression and purification

2025-06-05T13:39:22Z

ESO wikiadmin: /* Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26 */

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
[[File:SB20.png|right|frameless|328x328px]]
* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==

* Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
[[File:SBA 21.png|right|frameless|

*

]]

* Dialysis performed by SF
* Mix samples with 2x SDS loading buffer

* Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

* Perform SDS-Page at 80V/120V

* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==

* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)
* The column is hung upside down
* Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)
* Remove the tube from the end of the column and insert column the right way round
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL
* Prepare 6 Schnappdeckelröhrchen
* Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)
* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here
* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1
* [[File:SBA 24.png|right|frameless|389x389px]]Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2
* Wash column with elution buffer 2 for 15 min at 0.5 mL/min
* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page
* Run SDS-Page at 80V /120 V
* Stain gel with coomassie for 45 min at rt after short warming in microwave
* Destain until bands are visible

''Cleaning of Column after use:''

* wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)
* wash 20 min 1 mL/min with Wash buffer (20 mL)
* wash 20 mL 1 mL/min with H2O (degassed)
* wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==

* Mix samples with 2x SDS loading buffer
* Boil samples 95°C for 5 min
* Load onto a 12.5% acrylamide gel
* Run at 80 V/120 V
* Coomassie stain after short heating in microwave for 40 min
* Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)

o Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate

o Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)

o Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.

o Measure absorbance at 660 nm

o Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer

File:SBA 24.png

2025-06-05T13:38:54Z

ESO wikiadmin:

SBA 24

Protein expression and purification

2025-06-05T13:37:29Z

ESO wikiadmin:

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
[[File:SB20.png|right|frameless|328x328px]]
* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==

* Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
[[File:SBA 21.png|right|frameless|

*

]]

* Dialysis performed by SF
* Mix samples with 2x SDS loading buffer

* Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

* Perform SDS-Page at 80V/120V

* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==

* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)
* The column is hung upside down
* Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)
* Remove the tube from the end of the column and insert column the right way round
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL
* Prepare 6 Schnappdeckelröhrchen
* Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)
* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here
* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1
* Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2
* Wash column with elution buffer 2 for 15 min at 0.5 mL/min
* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page
* Run SDS-Page at 80V /120 V
* Stain gel with coomassie for 45 min at rt after short warming in microwave
* Destain until bands are visible

''Cleaning of Column after use:''

* wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)
* wash 20 min 1 mL/min with Wash buffer (20 mL)
* wash 20 mL 1 mL/min with H2O (degassed)
* wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
- Mix samples with 2x SDS loading buffer

- Boil samples 95°C for 5 min

- Load onto a 12.5% acrylamide gel

- Run at 80 V/120 V

- Coomassie stain after short heating in microwave for 40 min

- Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)

o Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate

o Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)

o Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.

o Measure absorbance at 660 nm

o Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer

Protein expression and purification

2025-06-05T13:37:00Z

ESO wikiadmin:

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
[[File:SB20.png|right|frameless|328x328px]]
* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==

* Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
[[File:SBA 21.png|right|frameless|

*

]]

* Dialysis performed by SF
* Mix samples with 2x SDS loading buffer

* Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

* Perform SDS-Page at 80V/120V

* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==

* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)
* The column is hung upside down
* Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)
* Remove the tube from the end of the column and insert column the right way round
* Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL
* Prepare 6 Schnappdeckelröhrchen
* Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)
* Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here
* Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1
* Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2
* Wash column with elution buffer 2 for 15 min at 0.5 mL/min
* Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page
* Run SDS-Page at 80V /120 V
* Stain gel with coomassie for 45 min at rt after short warming in microwave
* Destain until bands are visible

''Cleaning of Column after use:''

- wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)

- wash 20 min 1 mL/min with Wash buffer (20 mL)

- wash 20 mL 1 mL/min with H2O (degassed)

- Wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
- Mix samples with 2x SDS loading buffer

- Boil samples 95°C for 5 min

- Load onto a 12.5% acrylamide gel

- Run at 80 V/120 V

- Coomassie stain after short heating in microwave for 40 min

- Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)

o Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate

o Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)

o Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.

o Measure absorbance at 660 nm

o Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer

Protein expression and purification

2025-06-05T13:35:33Z

ESO wikiadmin:

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
[[File:SB20.png|right|frameless|328x328px]]
* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==

* Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)
* Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
[[File:SBA 21.png|right|frameless|

*

]]

* Dialysis performed by SF
* Mix samples with 2x SDS loading buffer

* Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

* Perform SDS-Page at 80V/120V

* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
- Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)

- The column is hung upside down

- Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)

- Remove the tube from the end of the column and insert column the right way round

- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL

- Prepare 6 Schnappdeckelröhrchen

- Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)

- Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here

- Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1

- Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2

- Wash column with elution buffer 2 for 15 min at 0.5 mL/min

- Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page

- Run SDS-Page at 80V /120 V

- Stain gel with coomassie for 45 min at rt after short warming in microwave

- Destain until bands are visible

''Cleaning of Column after use:''

- wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)

- wash 20 min 1 mL/min with Wash buffer (20 mL)

- wash 20 mL 1 mL/min with H2O (degassed)

- Wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
- Mix samples with 2x SDS loading buffer

- Boil samples 95°C for 5 min

- Load onto a 12.5% acrylamide gel

- Run at 80 V/120 V

- Coomassie stain after short heating in microwave for 40 min

- Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)

o Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate

o Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)

o Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.

o Measure absorbance at 660 nm

o Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer

File:SBA 21.png

2025-06-05T13:34:34Z

ESO wikiadmin:

SBA 21

Protein expression and purification

2025-06-05T13:32:54Z

ESO wikiadmin:

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
[[File:SB20.png|right|frameless|328x328px]]
* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==
- Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
- Dialysis performed by SF

- Mix samples with 2x SDS loading buffer

- Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

- Perform SDS-Page at 80V/120V

- Coomassie stain gel after short heating in the microwave for 30 min at rt

- Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
- Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)

- The column is hung upside down

- Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)

- Remove the tube from the end of the column and insert column the right way round

- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL

- Prepare 6 Schnappdeckelröhrchen

- Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)

- Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here

- Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1

- Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2

- Wash column with elution buffer 2 for 15 min at 0.5 mL/min

- Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page

- Run SDS-Page at 80V /120 V

- Stain gel with coomassie for 45 min at rt after short warming in microwave

- Destain until bands are visible

''Cleaning of Column after use:''

- wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)

- wash 20 min 1 mL/min with Wash buffer (20 mL)

- wash 20 mL 1 mL/min with H2O (degassed)

- Wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
- Mix samples with 2x SDS loading buffer

- Boil samples 95°C for 5 min

- Load onto a 12.5% acrylamide gel

- Run at 80 V/120 V

- Coomassie stain after short heating in microwave for 40 min

- Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)

o Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate

o Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)

o Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.

o Measure absorbance at 660 nm

o Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer

Protein expression and purification

2025-06-05T13:31:57Z

ESO wikiadmin:

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
[[File:SB20.png|right|frameless]]
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==

* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==
- Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
- Dialysis performed by SF

- Mix samples with 2x SDS loading buffer

- Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

- Perform SDS-Page at 80V/120V

- Coomassie stain gel after short heating in the microwave for 30 min at rt

- Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
- Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)

- The column is hung upside down

- Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)

- Remove the tube from the end of the column and insert column the right way round

- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL

- Prepare 6 Schnappdeckelröhrchen

- Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)

- Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here

- Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1

- Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2

- Wash column with elution buffer 2 for 15 min at 0.5 mL/min

- Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page

- Run SDS-Page at 80V /120 V

- Stain gel with coomassie for 45 min at rt after short warming in microwave

- Destain until bands are visible

''Cleaning of Column after use:''

- wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)

- wash 20 min 1 mL/min with Wash buffer (20 mL)

- wash 20 mL 1 mL/min with H2O (degassed)

- Wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
- Mix samples with 2x SDS loading buffer

- Boil samples 95°C for 5 min

- Load onto a 12.5% acrylamide gel

- Run at 80 V/120 V

- Coomassie stain after short heating in microwave for 40 min

- Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)

o Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate

o Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)

o Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.

o Measure absorbance at 660 nm

o Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer

File:SB20.png

2025-06-05T13:31:08Z

ESO wikiadmin:

SBA20

Protein expression and purification

2025-06-05T13:30:24Z

ESO wikiadmin:

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==

* '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength
* One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C
* 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7
* When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction
* 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h
* 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.
* Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)
* Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant
* Transfer these 15 mL of resuspension into a 50 mL Falcon
* Centrifuge at 4700 rpm for 15 min
* Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

* Thaw bacterial pellet at 4°C
* Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

* Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween
* Centrifuge for 30 min at 18000 rpm
* Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

* COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min
* Apply supernatant post centrifugation with 0.5 mL/min
* Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C
* Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

* Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==

* Purification of recombinant PPM1F via HisSUMO tag conducted by SF
* Mix samples with 2x SDS loading buffer
* Load samples onto 12.5% Acrylamide gel
* Perform SDS-Page at 80V/120V
* Coomassie stain gel after short heating in the microwave for 30 min at rt
* Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==
- Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
- Dialysis performed by SF

- Mix samples with 2x SDS loading buffer

- Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

- Perform SDS-Page at 80V/120V

- Coomassie stain gel after short heating in the microwave for 30 min at rt

- Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
- Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)

- The column is hung upside down

- Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)

- Remove the tube from the end of the column and insert column the right way round

- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL

- Prepare 6 Schnappdeckelröhrchen

- Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)

- Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here

- Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1

- Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2

- Wash column with elution buffer 2 for 15 min at 0.5 mL/min

- Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page

- Run SDS-Page at 80V /120 V

- Stain gel with coomassie for 45 min at rt after short warming in microwave

- Destain until bands are visible

''Cleaning of Column after use:''

- wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)

- wash 20 min 1 mL/min with Wash buffer (20 mL)

- wash 20 mL 1 mL/min with H2O (degassed)

- Wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
- Mix samples with 2x SDS loading buffer

- Boil samples 95°C for 5 min

- Load onto a 12.5% acrylamide gel

- Run at 80 V/120 V

- Coomassie stain after short heating in microwave for 40 min

- Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)

o Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate

o Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)

o Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.

o Measure absorbance at 660 nm

o Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer

Protein expression and purification

2025-06-05T13:27:01Z

ESO wikiadmin: Created page with "== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' == - '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength - One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C - 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7 - When OD is reach..."

== '''Expression of HisSUMO-hPOPX2 (SAK) – Part 1/2''' ==
- '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength

- One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C

- 7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7

- When OD is reached, Erlenmayers were shortly transferred onto ice for temperature reduction

- 0.5 ml is removed (sample before induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.

- Bacterial cultures were induced with 0.5 mM IPTG and subsequently incubated at 20°C upon shaking (160 rpm) for 4 h

- 0.5 ml is removed (sample after induction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 μl 2xSDS sample buffer and heated for 5 min at 90°C.

- Remaining cells are centrifuged for 30 min at RT with 4700 rpm (be careful to only use the old centrifugation flasks with the old centrifuge under the incubator and do not use those with the big centrifuge in front of ZK)

- Carefully remove supernatant and resuspend pellet in remaining approx. 15 mL supernatant

- Transfer these 15 mL of resuspension into a 50 mL Falcon

- Centrifuge at 4700 rpm for 15 min

- Aspirate supernatant and freeze pellet at -80°C

== '''PPM1F Expression in Bacteria - Recombinant hPOPX2 (PPM1F) purification (conducted by SF) – Part 2/2''' ==
Lysis of bacteria:

- Thaw bacterial pellet at 4°C

- Resuspend in 15 mL Buffer A with protease inhibitors (1 µM PMSF, 10 µg/mL aprotinin, 10 µg/mL pefablock, 5 µg/mL Leupeptin)

a. '''BUFFER A:''' 50 mM NaPhosphat, 1 M NaCl pH 8.0

- Sonificate: 3 x 2 min 4°C with approx.. 5 min break inbetween

- Centrifuge for 30 min at 18000 rpm

- Filtrate supernatant through a 0.2 µm pore size filter and apply onto column

Purification via column:

- COLUMN: HisTrapFFcrude 1 mL (Pharmacia), equilibrate with 20 mL 50 mM NaPhosphate buffer, 1 M NaCl, pH 8.0 1 mL/min

- Apply supernatant post centrifugation with 0.5 mL/min

- Wash 60 min with buffer A + 10 mM Imidazol 1 mL/min, 4°C

- Elution: Gradient 0-50% Buffer B for 40 min; 20 min 50% Buffer B; collect fractions with 3 min/fraction  ~ 3 mL per fraction

a. '''BUFFER B:''' 50 mM NaPhosphat, 0.5 M NaCl, 0.5 M Imidazol pH 8.0

- Wash column for 20 min with 100% buffer B

== '''Gel-Electrophoresis post purification of recombinant hPOPX2 (PPM1F)– Gel #20''' ==
- Purification of recombinant PPM1F via HisSUMO tag conducted by SF

- Mix samples with 2x SDS loading buffer

- Load samples onto 12.5% Acrylamide gel

- Perform SDS-Page at 80V/120V

- Coomassie stain gel after short heating in the microwave for 30 min at rt

- Destain until bands are visible

== '''PPM1F Expression in Bacteria – Dialysis (conductec by SF)''' ==
- Dialysis in 1.5 L o/n against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Dialysis in 1.5 L for 4 h against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Dialysis in 1.5 L for 4 h 30 min against 20 mM Tris pH8, 150 mM NaCl, 1.5 mM MgCl2 (no glycerin!)

- Observation: some precipitates form, centrifuge 3000 rpm 10 min at 4°C

== '''Quantification Gel for recombinant hPOPX2 (PPM1F)– Gel #21''' ==
- Dialysis performed by SF

- Mix samples with 2x SDS loading buffer

- Load 2.5 or 5 µL of sample onto a 12.5% Acrylamide Gel

- Perform SDS-Page at 80V/120V

- Coomassie stain gel after short heating in the microwave for 30 min at rt

- Destain until bands are visible

'''→''' Impurities mainly in the fallout fraction (“Pellet”); Supernatant contains approx.. 400 ng/µL HisSUMO-POPX2

== '''Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Part 1/2''' ==
- Incubate 5 mL of purified His-SUMO hPOPX2 (conc. 400 ng/µL) with 5 µL Ulp1 (conc. 2 mg/mL) → 1 µg of Ulp1 per 200 µg of sample

== '''Purification post Ulp1 treatment for His-SUMO tag cleavage of His-SUMO hPOPX2 – Gel #24 – Part 2/2''' ==
- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) Install tubing of column in the pump (the tube is inserted from the bottom side in)

- The column is hung upside down

- Pump water at 0.5 ml/min for about 10 min, fill tubing first, then connect to column (?)

- Remove the tube from the end of the column and insert column the right way round

- Prime column with wash buffer (50 mM Sodium Phosphate buffer pH 8 with 1 M NaCl) 0.5 mL/min, 15-20 mL

- Prepare 6 Schnappdeckelröhrchen

- Prepare 3 mL sample by addition of 1:50 elution buffer (50 Mm Sodium phosphate buffer pH 8, 0.5 M NaCl and 0.5 M imidazole) and 1:10 of 5 M NaCl-stock solution)

- Pump sample onto column, collect flow through (DF, Durchfluss) → hPOPX2 should be eluted here

- Start with Elutionbuffer 1 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 10 mM Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 1

- Switch to Elutionbuffer 2 (50 mM Sodium phosphate buffer pH 8, 1 M NaCl, 0.5M Imidazole): per Schnappdeckelröhrchen pump 0.5 mL/min for 6 min → 3 mL/fraction collected. Collect three fractions with Elutionbuffer 2

- Wash column with elution buffer 2 for 15 min at 0.5 mL/min

- Mix 20 µL of sample with 20 µL 2x SDS, boil 5 min at 95°C and apply 20 µL to 12.5% SDS-Page

- Run SDS-Page at 80V /120 V

- Stain gel with coomassie for 45 min at rt after short warming in microwave

- Destain until bands are visible

''Cleaning of Column after use:''

- wash 10 min 1 mL/min with Elutionbuffer 2 (10 mL)

- wash 20 min 1 mL/min with Wash buffer (20 mL)

- wash 20 mL 1 mL/min with H2O (degassed)

- Wash 20 mL 20% EtOH

== '''Quantification Gel for recombinant hPOPX2 (PPM1F) after Ulp1 treatment and dialysis – Gel #26''' ==
- Mix samples with 2x SDS loading buffer

- Boil samples 95°C for 5 min

- Load onto a 12.5% acrylamide gel

- Run at 80 V/120 V

- Coomassie stain after short heating in microwave for 40 min

- Destain until bands become visible

== '''Concentration Determination of purified recombinant hPOPX2 (PPM1F)''' ==
- Determine Protein Conc. Via 660 nm Pierce Protein Assay Kit (ThermoScientific)

o Pipette 10 µL of BSA standard in PBS or 10 µL of blank or 10 µL of sample in triplicates into a 96 well plate

o Add 150 µL Pierce 660nm Protein Assay Reagent (storage: rt, JWK)

o Cover plate and mix on a plate shaker at medium speed for 1 minute. Incubate at room temperature for 5 minutes.

o Measure absorbance at 660 nm

o Perform standard curve and calculate the concentration of the protein sample

'''→''' Sample Concentration: 131,1027 ng/µL in 9 mL dialysis buffer

Lab Etiquette

2025-06-05T13:22:06Z

ESO wikiadmin:

'''Diese Seite ist auch auf [[Lab Etiquette (ENG)|Englisch 🇬🇧]] verfügbar.'''

== '''Laborzugang''' ==
Das Laborgebäude ML ist zwischen 6:30 und 20:30 Uhr an Werktagen geöffnet. Zugang zu den Laborräumen erhält man mittels RFID-Transponder, der es auch erlaubt, das Laborgebäude außerhalb der oben stehenden Öffnungszeiten zu betreten. Masterstudenten und Doktoranden erhalten einen persönlichen Transponder über die Schlüsselausgabe (Formular bei Petra Schnurr) und müssen den Transponder nach Beendigung ihrer Zeit als Mitarbeiter wieder abgeben. Für Bachlorstudenten und Hiwis gibt es nach Einarbeitung in Ausnahmefällen einen Leihtransponder für notwendige Arbeiten außerhalb der Öffnungszeiten. Der Leihtransponder befindet sich im Schlüsselkasten am allg. Computer-Arbeitsplatz. Die Entnahme des Leihtransponders ist in der Liste mit Datum und Kürzel zu vermerken.

== '''Arbeitszeiten''' ==
Nicht-wissenschaftliche Mitarbeiter und Hiwis sind verpflichtet, die Arbeitszeit zu erfassen. Hierzu steht den Mitarbeitern die Online-Webseite der Personalverwaltung zur Verfügung:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

Hiwis nutzen zur Zeiterfassung den Stundenzettel (erhältlich bei Petra Schnurr)

Wissenschaftliche Mitarbeiter sollen zu den Kernarbeitszeiten 9.00 – 12.00 und 14.00 – 16.00 anwesend sein. Eine Abwesenheit ist mit Christof abzusprechen und im Wandkalender im Eingangsbereich der Bürozeile zu vermerken.

== '''Krankheit''' ==
Im Krankheitsfall soll so bald wie möglich Christof und das Sekretariat per Telefon und/oder E-Mail benachrichtigt werden, damit Vertretung, etc. organisiert werden kann. Bei längerer Krankheit muss am dritten Tag der Abwesenheit eine ärztliche Krankmeldung/ein ärztliches Attest an petra.schnurr@uni-konstanz.de geschickt werden.

== '''Urlaub''' ==
Mitarbeiter beantragen möglichst frühzeitig (mindestens 1 Woche vorher) ihren Urlaub über das online Zeiterfassungssystem:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

Die Möglichkeit für längerfristigen Urlaub (> 2 Wochen) ist projektabhängig und soll mindestens 2 Monate vorher mit Christof abgesprochen werden. In der 2.Hälfte des Wintersemesters (Anfang Januar – Ende Februar) ist während des VTK Urlaub nur in Ausnahmefällen möglich.

Vor Urlaubsantritt muss immer die Kultur/Lagerung von wichtigen Klonen/Zellinien/Proben sichergestellt und eine Vertretung für den Labjob organisiert sein. Bachelor-/Masterstudenten sprechen eine mehrtägige Abwesenheit mit Ihrem/Ihrer BetreuerIn und Christof ab.

== '''Seminare/Projektbesprechungen''' ==
Montags vormittags (8.45 Uhr) ist eine allg. Laborbesprechung. Im Anschluß daran eine Porjektvorstellung einzelner Mitarbeiter, die am Notice-Board im Eingangsbereich angekündigt wird.

Mittwochs am Nachmittag von 15.15 Uhr – ca. 17.00 Uhr findet unser Journal-Club statt. Die jeweils behandelte Publikation wird in der Woche zuvor auf den Fileserver geladen bzw. als pdf-file an alle verschickt. Im 14-tägigen Turnus besprechen wir in diesem Seminar aktuelle neue Daten aller Mitarbeiter.

Die Teilnahme an diesen beiden Seminaren ist obligatorisch, der Seminarplan wird in der Regel auf ein viertel- bis ein halbes Jahr im voraus festgelegt.

Dienstags nachmittags und donnerstags zur Mittagszeit finden während der Vorlesungszeit die Seminare der Graduiertenschule KoRS-CB sowie das Fachbereichsseminar des FB Biologie statt. Die Vortragenden/Themen werden durch Aushang bekannt gegeben. Die Teilnahme ist fakultativ, aber wenn möglich sollte man sich die interessanten Vorträge nicht entgehen lassen.

Spezifische Projektbesprechungen finden nach Absprache mit Christof statt. Zu den Projektbesprechungen ist das Laborbuch als Gesprächsgrundlage mitzubringen.

== '''Namenskürzel/Beschriftungen''' ==
Jeder Mitarbeiter benutzt ein unverwechselbares Namenskürzel, mit dem alle Proben, Lösungen, etc. beschriftet werden müssen. Die Namenskürzel werden im Sekretariat vergeben und sind in einer Liste auf dem Fileserver abgelegt:

Fileserver_LSH/Vorlagen/Labor-Verzeichnisse/Namenskuerzel_AG Hauck_2025.doc

== '''Laborbuch''' ==
Jeder Mitarbeiter führt ein Laborbuch, das im Sekretariat ausgegeben wird und in dem alle Arbeiten in chronologischer Reihenfolge dokumentiert werden. Muster für die Dokumentation von verschiedenen Experimenten sind als pdf-file auf dem Fileserver abgelegt:

Fileserver_LSH/Vorlagen/Labor-Protokollierung/

* Gentamicin-Assay: „Steffi“
* PCR: „''PCR-based-LIC-cloning-protocol-CRH.pdf''“
* Zelltransfektion: „''HEKcell-transfection-Protocol-JWK.pdf''“
* In vitro Kinase Assay: “Erik”
* [[Immunoprecipitation|Immunopräzipitation]] (MHE)
* [[Protein expression and purification]]: “Sarah”
* Lentivirus production: “Marlene”
* Bacterial growth curve: “Ann-Kathrin”

Die Seiten des Laborbuchs werden durchnumeriert und auf den ersten beiden Seiten wird ein Inhaltsverzeichnis angelegt.

Das Laborbuch gehört dem Lehrstuhl Zellbiologie und verbleibt nach dem Ausscheiden im Besitz des Lehrstuhls. Aus dem Laborbuch dürfen keine Seiten entfernt werden. Originalausdrucke, Kopien von Gelen, Röntgenfilme, etc. werden beschriftet und dem Laborbuch beigelegt, indem sie zu den entsprechenden Experimenten geheftet oder geklebt werden. Die elektronischen Originaldaten werden auf dem Fileserver in einem eigenen User-Verzeichnis gespeichert (s. Kapitel Daten Archivierung).

Für die meisten Experimente gibt es bereits Labor-Protokolle, die in unserem VTK-Skript ausführlich beschrieben sind und auf die im Laborbuch verwiesen werden kann. Das VTK-Skript findet sich auf dem Fileserver:

''Fileserver_LSH/Vorlagen/Labor-Protokollierung/Skript-Vertiefungskurs-2025.doc''

Die verwendeten Reagenzien und Lösungen sind ebenfalls entweder in der Rezepte-Datei beschrieben: ''Fileserver_LSH/Rezepte/Rezepte-2025.doc'' oder sind in der Chemikalienliste zu finden:

''Fileserver_LSH/Chemikalienliste/Chemikalienliste 2025 Gruppe Hauck.xls''

Nur bei Abweichen vom existierenden Protokoll sind besondere Vermerke im Laborbuch notwendig.

Bei mikroskopischen Arbeiten, durchflußzytometrischen Analysen oder ähnlichen Arbeiten mit Ergebnissen in elektronischer Form wird ein Probenprotokoll angefertigt (s. Fileserver_LSH/Vorlagen/Labor-Protokollierung/Protokoll-Mikroskop-FACS.doc“) und dem Experiment beigeheftet.

Bei Projektbesprechungen dient das Laborbuch als Grundlage für die Diskussion der Ergebnisse und ist deshalb unbedingt mitzubringen.

== '''Daten-Archivierung''' ==
Standardexperimente sollen anhand der oben angeführten Laborprotokolle durchgeführt werden, Abweichungen sind im Laborbuch festzuhalten. Die im Rahmen der Experimente erhaltenen Rohdaten (z.B. Mikroskopiebilder, Gelbilder, FACS-Daten, VarioScan-Daten) sowie die daraus hergestellten abgeleiteten Abbildungen (Graphiken, berbeiteten Mikroskopiebilder, etc.) sind auf dem Daten-Fileserver „Fileserver_LSH_Data/00_User/„ in einem separaten Nutzerfolder zu speichern. Nur dort erfolgt eine automatische (tägliche) Sicherung, so dass keine Daten verloren gehen können.

Jede elektronische Datendatei trägt im Namen das Datum, das Namenskürzel und das Experiment (The raw data files are named according to the following scheme):

'''Jahr-Monat-Tag_Namenskürzel_Experiment / year-month-day_initials_experiment'''

z.B. /e.g.: 2025-01-01_CRH_Adhesion assay

- the digital version/raw files should be on the file server with the date that can also be found in the lab book

- a printed version of the evaluated data should be attached in the lab book

For later use in publications it is important that we all stick to the same folder structure and naming of files. An example of such a user folder is given on the Fileserver and depicted on the next page:

Fileserver_LSH_Data/00_User/xUE_Universal_Example/

'''Data'''

'' Constructs''

SuperSH2 agarose gels, sequencing results, plasmid map, etc.

'' Western Blots''

'' Immunofluorescence staining''

'' Flow Cytometry''

'' ELISA/SEAP-Assays''

''' .'''

''' .'''

'''Literature'''

'''Presentations'''

'''Thesis:''' for your thesis: for '''every''' figure in your thesis create a subfolder named with the Figure-Number and include in each subfolder both: the final figure as pdf- and illustrator-file and the '''used raw data''' (subdivided in panels A, B, etc.). The name of the raw data file should remain the same as it is found in your DATA-folder and lab book)
[[File:Labetiquette fileserver.jpg|center|thumb|700x700px|'''Fig. 1:''' Example of the user folder structure on the Fileserver_LSH_Data of user UE_Universal_Example.]]

== '''Laborarbeitsplatz''' ==
In den Laboren gibt es allgemeine Laborarbeitsplätze (z.B. in den beiden Zellkulturlaboren, im Infektionsraum oder der Bereich mit Proteingel-Elektrophorese) sowie private Arbeitsplätze, die zugewiesen sind. Arbeiten auf den privaten Arbeitsplätzen anderer soll nur nach Rücksprache erfolgen. Jeder ist für die Sauberhaltung seines Arbeitsplatzes, der dazugehörigen Regale und Schubladen selbst verantwortlich. Die Arbeitsfläche wird mindestens einmal pro Woche abgewischt und desinfiziert, die Schubladen/Regale sind regelmäßig abzustauben und ordentlich zu halten.

Zusätzlich hat jeder einen Lagerplatz in einem Kühlschrank bei 4°C, in einem Gefrierschrank/-fach bei -20°C und kann Proben in beschrifteten Boxen im -80°C-Schrank in Raum ML lagern. Für die Lagerung stehen Kunststoffboxen mit Deckel zur Verfügung, offenen Ständer (speziell für Eppendorfgefäße) sollen nur für Kurzzeit-Aufbewahrung (1 Tag) verwendet werden. Alle Proben (Eppis, Agarplatten, etc.) müssen mit Kürzel, Inhaltsbezeichnung und Datum beschriftet sein. Alle Lagerplätze sollen regelmäßig (mindestens 1x je Monat) aufgeräumt/aktualisiert werden. Bei Lagerung im -80°C Schrank ist für jede Box ein Datenblatt in den -80°C-Ordner abzulegen. Eine Vorlage für das Datenblatt findet sich auf dem Fileserver:

''Fileserver_LSH/Vorlagen/Labor-Protokollierung/'' ''-80C Box-Vorlage.doc''

Das Öffnen von Tiefgefrierschränken bitte auf ein Minimum reduzieren (da sonst Vereisung und Fehlfunktion). Deshalb bereits VOR dem Öffnen überlegen, was entnommen werden soll und wo es zu finden ist (s. Datenblatt!!!). Allgemeine Laborarbeitsplätze sind nach jeder Benutzung zu säubern (z.B. sterile Werkbank, Proteingel-Elektrophorese, Abzug, Waagen, Geldoku) und alle verwendeten Lösungen, Apparaturen, etc. müssen wieder aufgeräumt werden.

== '''Lab-Jobs''' ==
Jeder Mitarbeiter erhält einen Labjob, den er für die Allgemeinheit wahrnimmt. Der Lab-Job kann die Pflege einer allgemeinen Sammlung, die Verantwortung für ein Gerät oder die Aufsicht und Organisation eines Labors beinhalten. Darüber hinaus wird die Herstellung von Lösungen/Puffern etc. für die Allgemeinheit über Lab-Jobs. Die jeweils Verantwortlichen sind auf einer Liste am am Noticeboard am Eingang aufgeführt. Die Raum-Verantwortlichen organisieren auch die regelmäßige Pflege/Putzpläne für allgemeine Geräte wie sterile Werkbänke, Inkubatoren, etc.

== '''Schließdienst''' ==
Der abendliche Schließdienst, der dafür sorgt, dass Geräte wie sterile Werkbänke, Vakkumpumpen, Geldoku oder die Propangasleitung ausgeschaltet sind, wechselt im wöchentlichen Turnus unter den wissenschaftlichen Mitarbeitern und Masterstudenten. Eine Liste für den abendlichen Kontrollgang des Schließdienstes hängt am Noticeboard am Eingang.

== '''Computernutzung''' ==
Im Seminarraum befindet sich ein Computer zur allgemeinen Nutzung. Bitte jederzeit die Nutzung für dienstliche Zwecke ermöglichen. Private PCs/Notebooks können an das Netzwerk angeschlossen werden. Voraussetzung ist ein aktueller Virenscanner und die Aktivierung einer Firewall. Den Zugang zu den Netzwerk-Speichern des Lehrstuhls im Rechenzentrum (Fileserver_LSH und Fileserver_LSH_Data) richtet Christof ein. Auf dem Fileserver_LSH_Data/02_SOFTWARE stehen verschiedene Software-Programme zur Verfügung, darunter Adobe Photoshop CS4, Adobe Acrobat Pro9, Adobe Illustrator CS4, MS Office 2016, Clone Manager 9, Endnote X9, SigmaStat 4.0, Leica LAS, Biorad QuantityOne, VarioScan SkanIt. Um den problemlosen Austausch der Daten innerhalb des Labors zu gewährleisten, müssen die oben angegebenen Versionen dieser Programme für alle dienstlichen Zwecke verwendet werden bzw. die Daten in einer mit diesen Programmversionen kompatiblen Form gespeichert werden.

== '''Die Mitarbeit jedes Einzelnen ist gefragt''' ==
Jeder Mitarbeiter kann durch wenig Aufwand dazu beitragen, dass sich die Zusatzarbeiten im Labor für Alle minimieren. So soll jeder durch vorausschauende Planung den anfallenden Verbrauch/Abfall/Aufräumaufwand möglichst gering halten und auch bei der Entsorgung von Abfällen mithelfen. Bitte Autoklaviersäcke nicht bis zum „Überquellen“ füllen, sondern rechtzeitig verschließen, in der Wanne deponieren und neuen Autoklaviersack in den entsprechenden Müllbehälter geben.

Jeder ist auch selbst für das Befüllen der Boxen mit Pipettenspitzen und das Auffüllen von Eppis verantwortlich. Autoklaviergut wird in der Plastikkiste gesammelt und von Claudia/Susanne für alle autoklaviert.

Zum verträglichen Umgang gehört auch das Verhalten und die sauberkeit in der Teeküche und im Sozialraum. Wenn ich im Verlaufe von 1 Monat nie die Spülmaschine ein- oder ausgeräumt oder die Kaffeemaschine aufgefüllt habe, dann habe ich mich sehr wahrscheinlich auf Kosten von Anderen am sauberen Geschirr bedient.

== '''Chemikalien/Lösungen''' ==
Viele Lösungen werden als Lab-Job angesetzt und entweder als Stocklösung an festgelegten Plätzen für Alle zugänglich aufbewahrt oder als aliquotierte Lösung bereitgestellt, wobei man sich bei Bedarf ein Aliquot an den eigenen Arbeitsplatz mitnimmt und dort für die weitere Verwendung aufbewahrt. Weitere Lösungen für den eigenen Bedarf sind selbst anzusetzen (bitte die benötigten Mengen im Voraus abschätzen und nicht Liter-weise Lösungen ansetzen, wenn man für zwei Experimente insgesamt nur 10 ml braucht) und korrekt beschriftet (Kürzel, Datum, Inhalt: Bezeichnung und Konzentration; Gefahrensymbol) am eigenen Arbeitsplatz aufzubewahren. Lösungen und Aliquots am Arbeitsplatz der Kollegen NUR nach Rücksprache benutzen. Stocklösungen, aliquotierte Lösungen und die jeweiligen Verantwortlichen sind auf der Liste am Schwarzen Brett „Lab-Jobs“ aufgeführt. Für das Ansetzen von Lösungen gibt es ein Rezeptbuch, das als Datei auf dem Fileserver bereit liegt:

Fileserver_LSH//Rezepte/ Rezepte-2025.doc

== '''Allgemeine Sammlungen''' ==
Der Lehrstuhl unterhält verschiedene Sammlungen, die eine wichtige Ressource für Alle darstellen und mit denen deshalb besonders umsichtig und verantwortlich umgegangen werden muss.

Stammsammlung: umfasst alle Bakterienstämme, die nach Nummern abgelegt sind. Lagerung bei -70°C in Truhe auf Ebene 5 sowie in Raum ML6 :

– nichtpathogene Bakterien (=gleichzeitig Repositorium aller hergestellten Plasmide)

– pathogene Bakterien und Mutanten (Schlüssel dafür bei Christof)

Die Stammsammlung ist auch als MS Access-Datei vorhanden. Jeder neu hergestellte oder von extern erhaltene Bakterienstamm erhält eine neue Stammnummer und wird in der Stammsammlung eingefroren sowie elektronisch erfasst. Die Verteilung der Nummern erfolgt über Christof. Zugang zur Stammsammlung haben nur Susanne und Christof.

Plasmidsammlung: umfasst alle isolierten Plasmide aus den nichtpathogenen Bakterienstämmen der Stammsammlung. Die Numerierung ist identisch mit der Stammsammlung (Lagerung im allg. Kühlschrank bei 4°C)

Zellsammlung: umfasst Stocks aller Zellinien und primärer Zellen. Werden im flüssigen Stickstoff in der Medienküche aufbewahrt. Die Zellsammlung wird von Susanne verwaltet.

Virensammlung: umfasst alle lentiviralen, adenoviralen, AAV-Partikel und Bakteriophagen. Werden bei -70°C aufbewahrt. Access-Datei!

Oligosammlung: umfasst alle Oligos, die für Klonierungen, PCRs, etc. bestellt wurden. Die Oligos werden als 10 mM Stammlösung in TE-Puffer bei -20°C in der allgemeinen Sammlung, nicht in privaten Gefrierschränken, aufbewahrt. Bestellung neuer Oligos und Ablage der Informationen erfolgt über Jan Kuiper.

Antikörper-Sammlung: Sammlung aller Primärantikörper (kommerziell oder selbst hergestellt) und Sekundärantikörper (Enzym- oder Fluoreszenz-konjugiert). Datenblätter werden im Ordner „Antikörper“ gesammelt. Claudia Hentschel organisiert die Antikörper-Sammlung.

Protein-Sammlung: Hier werden alle rekombinanten, gereinigten Proteine erfasst.

Inhibitor-Sammlung: Hier werden Informationen zu allen pharmakologischen Inhibitoren und die Konzentration, das Lösungsmittel und der Aufbewahrungsort von Stammlösungen abgelegt. _______________ organisiert die Inhibitor-Sammlung.

Enzym-Sammlung: umfasst alle kommerziellen und selbst hergestellten (DpnI) Restriktionsenzyme, die bei -20°C gelagert werden. Entnahme von Enzymen aus den Vorratsgefäßen nur mit FILTERSPITZEN!

Die Enzyme dürfen nur für eine möglichst kurze Zeitspanne (1-2 Minuten) aus dem Gefrierschrank entnommen werden (d.h. direkt vor Gebrauch) und werden während dieser kurzen Zeit in einem auf -20°C-gekühlten Paraffinblock gelagert.

''Ordner mit Dokumentation zu allen Sammlungen finden sich in den Regalen am allgemeinen Arbeitsplatz im Bürobereich.''

== '''Kompetente Zellen''' ==
Aliquots kompetenter Bakterien werden bei -80°C gelagert. Je Aliquot sind 200 µl weggefroren, ausreichend für 2 Transformationen. Verschiedene Stämme stehen für unterschiedliche Zwecke zur Verfügung. Der Standardklonierungsstamm ist NovaBlue. Bei der Entnahme möglichst zügig vorgehen, Erwärmung der Aliquots führt zu verringerter Kompetenz, nicht mehr Aliquots entnehmen als tatsächlich gebraucht werden.

== '''Enzyme''' ==
Polymerasen, Restriktionsenzyme, etc. werden bei -20°C in Boxen bzw. auf Paraffinblöcken gemeinsam mit den entsprechenden Puffern aufbewahrt. Diese Enzyme werden von Allen gemeinsam benutzt. Deshalb besonders reinlich mit den Enyzm-Stammlösungen umgehen und Verunreinigungen (Filterspitzen !!!) sowie Erwärmung vermeiden.

Bei der Entnahme von Enzymen, die Eppis mit der Stammlösung ausschließlich in einem auf -20°C-gekühlten Paraffinblöckchen zum Arbeitsplatz transportieren und darin zum Pipettieren belassen. Auch mit Paraffinblock möglichst das Enzym nur kurzzeitig aus dem -20°C Gefrierschrank entnehmen (< 2 Minuten) und sofort nach dem Pipettieren wieder zurückbringen. Beim Öffnen der Vorratsgefäße auf jeden Fall Kontakt der Deckelinnenseite mit der Haut vermeiden!!

== '''Bestellungen''' ==
Wenn allgemeine Chemikalien oder Einwegartikel zur Neige gehen, RECHTZEITIG (d.h. vor dem Aufbrauchen der letzten Packung oder dem Abpipettieren des letzten Mikroliters) Bescheid geben: entweder Anschreiben an die Tafel im Labor oder den Bestellern sagen. Projektspezifische Chemikalien/Antikörper etc. oder Laborutensilien/-geräte vor Bestellung mit Christof absprechen.

Für Oligobestellungen über Jan die Oligo-Informationen in die Bestell-Liste auf dem Fileserver eintragen: Fileserver_LSH:\Stammsammlung\Primer\01_PrimerOrdering_Primerbestellung

Andere on-line Bestellungen mit Christof absprechen.

Bestellung von Sequenzierungen über pre-paid labels von LGC oder GeneWiz. Bitte nicht mehr als 2 Sequenzierung pro neuem Konstrukt. Größeren Sequenzierbedarf mit Christof absprechen. Lieferungen von Reagenzien sollen dem Besteller übergeben werden. Besteller prüft auf Vollständigkeit (unter Nutzen des Lieferscheins) und bestätigt mit Datum und Unterschrift. Der Lieferschein und eventuell beiliegende Rechnungen werden im Sekretariat an Petra Schnurr weitergegeben. Bei Lieferung von Chemikalien, Antikörpern, Enzymen und anderen Reagenzien werden die Datenblätter mit einem Datum versehen und in den entsprechenden Ordnern abgelegt (s. „Allgemeine Sammlungen“). Auf den Datenblättern wird eventuell das Lösungsmittel, die Konzentration und eventuell abgefüllte Aliquots sowie der Lagerort vermerkt.

ALLE Chemikalien, Reagenzien, Enzyme, etc. werden an zugänglichen und bekannten Orten gelagert.

== '''Laborsicherheit''' ==
Siehe dazu [[All About Safety|Safety Guidelines]]

Testexpression in Pichia pastoris

2025-06-05T13:20:20Z

ESO wikiadmin:

{{Protokoll-Mix|titel=Materials|Inhalt=These reagents are important:
*YPD – medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))
*BMMY medium (yeast extract (10 g/L), peptone (20 g/L), yeast nitrogen base
(13,4 g/L), potassium phosphate pH 6.0 (0,1 M), biotin (0.4 mg/L), methanol (0,5 %))
*MeOH abs.
*Transformed Pichia clones
*50 mL falcons
*Spectrometer for OD measurments
*Cuvettes
*1,5 mL Eppis‘
*PBS
*4x SDS loading buffer}}

== Procedure ==

* Inoculate 5 mL YPD medium with one '''big''' colony
** in 50 ml falcon tube (secure lid with tape, don’t tighten lid)
* Grow o. n. 30 °C, 200 rpm
* Centrifuge at 2000 g, 5 min
* Resuspend the complete pellet in 5 mL BMMY medium
* Measure the OD
** Use a 1:100 dilution for measurement
* Start growth
** For starters you don’t need to dilute your suspension
** For optimization purposes you may want to adjust your starting OD
*** Recommended is a starting OD between OD 3 and OD 40
* Grow at 30 °C, 200 rpm
* Take samples of 200 µL and add 25 µL MeOH after 24 h, and 48 h
** Sample handling
*** Measure the OD (1:100 dilution recommended)
*** Centrifuge 5 min, 2000 rpm
*** Split into pellet and supernatant
*** Resuspend the supernatant in 200 µL PBS
*** Mix supernatant with 4x SDS loading buffer
*** Mix equivalent of OD 1 from pellets with 80 µL of 4x SDS loading buffer
* End the testexpression after 72 h, take samples

For optimization, you may want to try different methanol concentrations between 0,5 % and 1.5 % MeOH

Testexpression in Pichia pastoris

2025-06-05T13:17:50Z

ESO wikiadmin: Created page with " == Procedure == * Inoculate 5 mL YPD medium with one '''big''' colony ** in 50 ml falcon tube (secure lid with tape, don’t tighten lid) * Grow o. n. 30 °C, 200 rpm * Centrifuge at 2000 g, 5 min * Resuspend the complete pellet in 5 mL BMMY medium * Measure the OD ** Use a 1:100 dilution for measurement * Start growth ** For starters you don’t need to dilute your suspension ** For optimization purposes you may want to adjust your starting OD *** Recommended is a sta..."

== Procedure ==

* Inoculate 5 mL YPD medium with one '''big''' colony
** in 50 ml falcon tube (secure lid with tape, don’t tighten lid)
* Grow o. n. 30 °C, 200 rpm
* Centrifuge at 2000 g, 5 min
* Resuspend the complete pellet in 5 mL BMMY medium
* Measure the OD
** Use a 1:100 dilution for measurement
* Start growth
** For starters you don’t need to dilute your suspension
** For optimization purposes you may want to adjust your starting OD
*** Recommended is a starting OD between OD 3 and OD 40
* Grow at 30 °C, 200 rpm
* Take samples of 200 µL and add 25 µL MeOH after 24 h, and 48 h
** Sample handling
*** Measure the OD (1:100 dilution recommended)
*** Centrifuge 5 min, 2000 rpm
*** Split into pellet and supernatant
*** Resuspend the supernatant in 200 µL PBS
*** Mix supernatant with 4x SDS loading buffer
*** Mix equivalent of OD 1 from pellets with 80 µL of 4x SDS loading buffer
* End the testexpression after 72 h, take samples

For optimization, you may want to try different methanol concentrations between 0,5 % and 1.5 % MeOH

Transformation of Pichia pastoris

2025-06-05T13:10:11Z

ESO wikiadmin:

{{Protokoll-Mix|Inhalt=- 150 µL competent Pichia Pastoris (- 80 °C) 
- Plasmid of interest 
- EcoRV restriction enzyme 
- Sodium Acetate (3 M) 
- EtOH abs.(ice cold, -20 °C) 
- 70% EtOH (ice cold, -20 °C) 
- Ice bath 
- Electroporation cuvette 
- Electroporation device 
- Sorbitol (1 M), ice cold, -20 °C 
- YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L)) 
- YPD agar plate with G418 (500 mg/L)|titel=Materials}}

== Procedure ==

* Linearize 20 µg of plasmid of interest with EcoRV
* Purify via EtOH precipitation:
** Adjust sample volume to 50 µL (MilliQ)
** Add 5 µL sodium acetate, vortex
** Add 125 µL ice cold EtOH abs.; Incubate on ice for 15 min
** Centrifuge 30 min, 14’000 rpm, 4 °C
** Discard the supernatant
** Wash the pellet with 500 µL colt EtOH 70 %
** Centrifuge 15 min, 14’000 rpm, 4 °C
** Remove the supernatant, dry pellet at RT
** Dissolve in 15-25 µL MilliQ
* Thaw competent ''Pichia'' on ice
* Add 10 µg linearized plasmid
* Incubate on ice for 5 min
* Pipet into electroporation cuvette on ice
* Perform electroporation
** 1500 V, 125 µF, 200 Ω
** Adjust all settings
** Insert cuvette into apparate
** Press both red buttons until tone sounds
** Immediately add 500 µL ice cold sorbitol
** Add 500 µL YPD medium
* Grow 1 h at 30 °C, 200 rpm
* Plate 100 µL undiluted suspension and 1:10 dilution of YPD agar plates with G418; grow at 30°C
* Keep only the '''big''' colonies for testexpression

Transformation of Pichia pastoris

2025-06-05T13:09:50Z

ESO wikiadmin:

{{Protokoll-Mix|Inhalt=- 150 µL competent Pichia Pastoris (- 80 °C) 
- Plasmid of interest 
- EcoRV restriction enzyme 
- Sodium Acetate (3 M) 
- EtOH abs.(ice cold, -20 °C) 
- 70% EtOH (ice cold, -20 °C) 
- Ice bath 
- Electroporation cuvette 
- Electroporation device 
- Sorbitol (1 M), ice cold, -20 °C 
- YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L)) 
- YPD agar plate with G418 (500 mg/L)}}

== Procedure ==

* Linearize 20 µg of plasmid of interest with EcoRV
* Purify via EtOH precipitation:
** Adjust sample volume to 50 µL (MilliQ)
** Add 5 µL sodium acetate, vortex
** Add 125 µL ice cold EtOH abs.; Incubate on ice for 15 min
** Centrifuge 30 min, 14’000 rpm, 4 °C
** Discard the supernatant
** Wash the pellet with 500 µL colt EtOH 70 %
** Centrifuge 15 min, 14’000 rpm, 4 °C
** Remove the supernatant, dry pellet at RT
** Dissolve in 15-25 µL MilliQ
* Thaw competent ''Pichia'' on ice
* Add 10 µg linearized plasmid
* Incubate on ice for 5 min
* Pipet into electroporation cuvette on ice
* Perform electroporation
** 1500 V, 125 µF, 200 Ω
** Adjust all settings
** Insert cuvette into apparate
** Press both red buttons until tone sounds
** Immediately add 500 µL ice cold sorbitol
** Add 500 µL YPD medium
* Grow 1 h at 30 °C, 200 rpm
* Plate 100 µL undiluted suspension and 1:10 dilution of YPD agar plates with G418; grow at 30°C
* Keep only the '''big''' colonies for testexpression

Transformation of Pichia pastoris

2025-06-05T13:09:09Z

ESO wikiadmin:

{{Protokoll-Mix|Inhalt=- 150 µL competent Pichia Pastoris (- 80 °C) 
- Plasmid of interest 
- EcoRV restriction enzyme 
- Sodium Acetate (3 M)
- EtOH abs.(ice cold, -20 °C)
- 70% EtOH (ice cold, -20 °C)
- Ice bath
- Electroporation cuvette
- Electroporation device
- Sorbitol (1 M), ice cold, -20 °C
- YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))
- YPD agar plate with G418 (500 mg/L)}}

== Procedure ==

* Linearize 20 µg of plasmid of interest with EcoRV
* Purify via EtOH precipitation:
** Adjust sample volume to 50 µL (MilliQ)
** Add 5 µL sodium acetate, vortex
** Add 125 µL ice cold EtOH abs.; Incubate on ice for 15 min
** Centrifuge 30 min, 14’000 rpm, 4 °C
** Discard the supernatant
** Wash the pellet with 500 µL colt EtOH 70 %
** Centrifuge 15 min, 14’000 rpm, 4 °C
** Remove the supernatant, dry pellet at RT
** Dissolve in 15-25 µL MilliQ
* Thaw competent ''Pichia'' on ice
* Add 10 µg linearized plasmid
* Incubate on ice for 5 min
* Pipet into electroporation cuvette on ice
* Perform electroporation
** 1500 V, 125 µF, 200 Ω
** Adjust all settings
** Insert cuvette into apparate
** Press both red buttons until tone sounds
** Immediately add 500 µL ice cold sorbitol
** Add 500 µL YPD medium
* Grow 1 h at 30 °C, 200 rpm
* Plate 100 µL undiluted suspension and 1:10 dilution of YPD agar plates with G418; grow at 30°C
* Keep only the '''big''' colonies for testexpression

Transformation of Pichia pastoris

2025-06-05T13:07:54Z

ESO wikiadmin: Created page with " == Procedure == * Linearize 20 µg of plasmid of interest with EcoRV * Purify via EtOH precipitation: ** Adjust sample volume to 50 µL (MilliQ) ** Add 5 µL sodium acetate, vortex ** Add 125 µL ice cold EtOH abs.; Incubate on ice for 15 min ** Centrifuge 30 min, 14’000 rpm, 4 °C ** Discard the supernatant ** Wash the pellet with 500 µL colt EtOH 70 % ** Centrifuge 15 min, 14’000 rpm, 4 °C ** Remove the supernatant, dry pellet at RT ** Dissolve in 15-25 µL Mil..."

== Procedure ==

* Linearize 20 µg of plasmid of interest with EcoRV
* Purify via EtOH precipitation:
** Adjust sample volume to 50 µL (MilliQ)
** Add 5 µL sodium acetate, vortex
** Add 125 µL ice cold EtOH abs.; Incubate on ice for 15 min
** Centrifuge 30 min, 14’000 rpm, 4 °C
** Discard the supernatant
** Wash the pellet with 500 µL colt EtOH 70 %
** Centrifuge 15 min, 14’000 rpm, 4 °C
** Remove the supernatant, dry pellet at RT
** Dissolve in 15-25 µL MilliQ
* Thaw competent ''Pichia'' on ice
* Add 10 µg linearized plasmid
* Incubate on ice for 5 min
* Pipet into electroporation cuvette on ice
* Perform electroporation
** 1500 V, 125 µF, 200 Ω
** Adjust all settings
** Insert cuvette into apparate
** Press both red buttons until tone sounds
** Immediately add 500 µL ice cold sorbitol
** Add 500 µL YPD medium
* Grow 1 h at 30 °C, 200 rpm
* Plate 100 µL undiluted suspension and 1:10 dilution of YPD agar plates with G418; grow at 30°C
* Keep only the '''big''' colonies for testexpression

Generation of competent Pichia pastoris

2025-06-05T13:05:42Z

ESO wikiadmin:

{{Protokoll-Mix|Inhalt=Wildtype Pichia Pastoris yJC100, grown on YPD agar plate
*YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))
*Transformation buffer (Tris (10 mM) pH 7,5, Sorbitol (0,6 M), LiAc (100mM), DTT (10 mM))
*Sorbitol (1M)
*Inoculation loop
*Erlenmeyer flask 500 mL
*Spectrometer for OD measurement
*50 mL falcons}}

== Procedure ==
* Inoculate 5 mL of YPD medium with one big colony
* Grow 8 h at 30 °C, 200 rpm
* Inoculate 100 mL YPD medium with 1 mL preculture
* Grow o.n. at 30 °C, 200 rpm
* Measure the OD
* Aliquot 15 x 108 cells, centrifuge 5 min, 1500 g
* Resuspend in 30 mL transformation buffer each
* Incubate at RT for 30 min
* Centrifuge 5 min, 4°C, 1500 g
* Resuspend in 5 mL Sorbitol each
* Repeat 3x
* Resuspend in 320 µL Sorbitol
* Freeze down in 150 µL aliquots
* Store at -80 °C

Generation of competent Pichia pastoris

2025-06-05T13:05:25Z

ESO wikiadmin:

{{Protokoll-Mix|Inhalt=Wildtype Pichia Pastoris yJC100, grown on YPD agar plate
*YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))
*Transformation buffer (Tris (10 mM) pH 7,5, Sorbitol (0,6 M), LiAc (100mM), DTT (10 mM))
*Sorbitol (1M)
*Inoculation loop
*Erlenmeyer flask 500 mL
*Spectrometer for OD measurement
*50 mL falcons}}

===== Procedure =====

* Inoculate 5 mL of YPD medium with one big colony
* Grow 8 h at 30 °C, 200 rpm
* Inoculate 100 mL YPD medium with 1 mL preculture
* Grow o.n. at 30 °C, 200 rpm
* Measure the OD
* Aliquot 15 x 108 cells, centrifuge 5 min, 1500 g
* Resuspend in 30 mL transformation buffer each
* Incubate at RT for 30 min
* Centrifuge 5 min, 4°C, 1500 g
* Resuspend in 5 mL Sorbitol each
* Repeat 3x
* Resuspend in 320 µL Sorbitol
* Freeze down in 150 µL aliquots
* Store at -80 °C

Generation of competent Pichia pastoris

2025-06-05T13:04:45Z

ESO wikiadmin: Created page with "{{Protokoll-Mix|Inhalt=Wildtype Pichia Pastoris yJC100, grown on YPD agar plate *YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L)) *Transformation buffer (Tris (10 mM) pH 7,5, Sorbitol (0,6 M), LiAc (100mM), DTT (10 mM)) *Sorbitol (1M) *Inoculation loop *Erlenmeyer flask 500 mL *Spectrometer for OD measurement *50 mL falcons}} ===== Procedure ===== - Inoculate 5 mL of YPD medium with one big colony - Grow 8 h at 30 °C, 200 rpm..."

{{Protokoll-Mix|Inhalt=Wildtype Pichia Pastoris yJC100, grown on YPD agar plate
*YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))
*Transformation buffer (Tris (10 mM) pH 7,5, Sorbitol (0,6 M), LiAc (100mM), DTT (10 mM))
*Sorbitol (1M)
*Inoculation loop
*Erlenmeyer flask 500 mL
*Spectrometer for OD measurement
*50 mL falcons}}

===== Procedure =====
- Inoculate 5 mL of YPD medium with one big colony

- Grow 8 h at 30 °C, 200 rpm

- Inoculate 100 mL YPD medium with 1 mL preculture

- Grow o.n. at 30 °C, 200 rpm

- Measure the OD

- Aliquot 15 x 108 cells, centrifuge 5 min, 1500 g

- Resuspend in 30 mL transformation buffer each

- Incubate at RT for 30 min

- Centrifuge 5 min, 4°C, 1500 g

- Resuspend in 5 mL Sorbitol each

- Repeat 3x

- Resuspend in 320 µL Sorbitol

- Freeze down in 150 µL aliquots

- Store at -80 °C

Purification of GST-fusion proteins

2025-06-05T13:03:29Z

ESO wikiadmin:

Purification of GST-fusion proteins

2025-06-05T13:03:16Z

ESO wikiadmin:

{{Protokoll-Mix|titel=Materials|Inhalt=<pre>
LB-Medium 10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0,
ad 1l A.bidest
Ampicillin 100μg/ml
IPTG 100mM Isopropylthiogalactosid
T-buffer 100mM Tris (pH 8.0), 5mM EDTA
Lysis buffer (200 ml): 10 ml Tris pH 8.0 [1M] → 50 mM
4 ml EDTA [0.5 M] → 10 mM
30 ml NaCl [1M] → 150 mM
40 ml Glycerol [50 %] → 10 %
0.5 ml Triton [10 %] → 0.025 %
add freshly à 20 ml:
50 μl DTT [1M] → 2.5 mM
20 μl PMSF [10 mg/ml]
20 μl Leupeptin [10 mg/ml]
20 μl Aprotinin [10 mg/ml]
20 μl Pefablock [10 mg/ml]
Lysozyme 1 mg/ml
Sepharose- solution,
Glutathione-sepharose-solution
PBS (1x) 24g NaCl, 0.6g KCl, 3.45g Na₂HPO₄ x 7 H₂O, 0.6 g
KH₂PO₄, pH 7.4, ad 1l mit A.bidest.
</pre>}}

== Procedure ==
The frozen bacteria pellets are thawed in 5 ml T-buffer on ice and resuspended. Afterwards the samples are incubated with 1 ml lysozyme for 30 min. The cells are broken through the addition of 20 ml lysis buffer and the treatment of ultrasonic sound (impulse 50%, 20sec, 3-5x) on ice water.

300 µl sepharose-solution is added to the suspension and incubated for 10 min with head-over-head rotation. Afterwards the lysate is centrifuged for 20 min at 16000 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.

<!DOCTYPE html>
<html lang="de">
<head>
<meta charset="UTF-8">
<meta name="viewport" content="width=device-width, initial-scale=1.0">
<title>Laboratory Recipes</title>
<style>
body {
font-family: monospace;
line-height: 1.4;
margin: 20px;
}
.recipe-section {
margin-bottom: 15px;
}
.ingredient-name {
display: inline-block;
width: 200px;
vertical-align: top;
}
.concentration {
display: inline-block;
width: 120px;
}
.arrow {
margin: 0 10px;
}
.final-conc {
display: inline-block;
}
.indent {
margin-left: 200px;
}
</style>
</head>
<body>

<div class="recipe-section">
LB-Medium
10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0, 
ad 1l A.bidest
</div>

<div class="recipe-section">
Ampicillin
100μg/ml
</div>

<div class="recipe-section">
IPTG
100mM Isopropylthiogalactosid
</div>

<div class="recipe-section">
T-buffer
100mM Tris (pH 8.0), 5mM EDTA
</div>

<div class="recipe-section">
Lysis buffer (200 ml):
<div class="indent">
10  ml  Tris pH 8.0 [1M]→50 mM 
4   ml  EDTA [0.5 M]→10 mM 
30  ml  NaCl [1M]→150 mM 
40  ml  Glycerol [50 %]→10 % 
0.5 ml  Triton [10 %]→0.025 % 
add freshly à 20 ml: 
50  μl  DTT [1M]→2.5 mM 
20  μl  PMSF [10 mg/ml] 
20  μl  Leupeptin [10 mg/ml] 
20  μl  Aprotinin [10 mg/ml] 
20  μl  Pefablock [10 mg/ml]
</div>
</div>

<div class="recipe-section">
Lysozyme
1 mg/ml
</div>

<div class="recipe-section">
Sepharose- solution, 
Glutathione-sepharose-solution
</div>

<div class="recipe-section">
PBS (1x)
24g NaCl, 0.6g KCl, 3.45g Na₂HPO₄ x 7 H₂O, 0.6 g 
KH₂PO₄, pH 7.4, ad 1l mit A.bidest.
</div>

</body>
</html>

Purification of GST-fusion proteins

2025-06-05T12:59:43Z

ESO wikiadmin:

{{Protokoll-Mix|titel=Materials|Inhalt=LB-Medium 10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0, ad 1l A.bidest
Ampicillin 100µg/ml
IPTG 100mM Isopropylthiogalactosid
T-buffer 100mM Tris (pH 8.0), 5mM EDTA
Lysis buffer (200 ml): 10 ml Tris pH 8.0 [1M]  50 mM
4 ml EDTA [0.5 M]  10 mM
30 ml NaCl [1M]  150 mM
40 ml Glycerol [50 %]  10 %
0.5 ml Triton [10 %]  0.025 %
add freshly à 20 ml:
50 µl DTT [1M]  2.5 mM
20 µl PMSF [10 mg/ml]
20 µl Leupeptin [10 mg/ml]
20 µl Aprotenin [10 mg/ml]
20 µl Pefablock [10 mg/ml]
Lysozyme 1 mg/ml
Sepharose- solution. Glutathione-sepharose-solution
PBS (1x) 24g NaCl, 0.6g KCl, 3.45g Na2HPO4 x 7 H2O, 0.6 g
KH2PO4, pH 7.4, ad 1l mit A.bidest.}}

== Procedure ==
The frozen bacteria pellets are thawed in 5 ml T-buffer on ice and resuspended. Afterwards the samples are incubated with 1 ml lysozyme for 30 min. The cells are broken through the addition of 20 ml lysis buffer and the treatment of ultrasonic sound (impulse 50%, 20sec, 3-5x) on ice water.

300 µl sepharose-solution is added to the suspension and incubated for 10 min with head-over-head rotation. Afterwards the lysate is centrifuged for 20 min at 16000 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.

<!DOCTYPE html>
<html lang="de">
<head>
<meta charset="UTF-8">
<meta name="viewport" content="width=device-width, initial-scale=1.0">
<title>Laboratory Recipes</title>
<style>
body {
font-family: monospace;
line-height: 1.4;
margin: 20px;
}
.recipe-section {
margin-bottom: 15px;
}
.ingredient-name {
display: inline-block;
width: 200px;
vertical-align: top;
}
.concentration {
display: inline-block;
width: 120px;
}
.arrow {
margin: 0 10px;
}
.final-conc {
display: inline-block;
}
.indent {
margin-left: 200px;
}
</style>
</head>
<body>

<div class="recipe-section">
LB-Medium
10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0, 
ad 1l A.bidest
</div>

<div class="recipe-section">
Ampicillin
100μg/ml
</div>

<div class="recipe-section">
IPTG
100mM Isopropylthiogalactosid
</div>

<div class="recipe-section">
T-buffer
100mM Tris (pH 8.0), 5mM EDTA
</div>

<div class="recipe-section">
Lysis buffer (200 ml):
<div class="indent">
10  ml  Tris pH 8.0 [1M]→50 mM 
4   ml  EDTA [0.5 M]→10 mM 
30  ml  NaCl [1M]→150 mM 
40  ml  Glycerol [50 %]→10 % 
0.5 ml  Triton [10 %]→0.025 % 
add freshly à 20 ml: 
50  μl  DTT [1M]→2.5 mM 
20  μl  PMSF [10 mg/ml] 
20  μl  Leupeptin [10 mg/ml] 
20  μl  Aprotinin [10 mg/ml] 
20  μl  Pefablock [10 mg/ml]
</div>
</div>

<div class="recipe-section">
Lysozyme
1 mg/ml
</div>

<div class="recipe-section">
Sepharose- solution, 
Glutathione-sepharose-solution
</div>

<div class="recipe-section">
PBS (1x)
24g NaCl, 0.6g KCl, 3.45g Na₂HPO₄ x 7 H₂O, 0.6 g 
KH₂PO₄, pH 7.4, ad 1l mit A.bidest.
</div>

</body>
</html>

Purification of GST-fusion proteins

2025-06-05T12:59:35Z

ESO wikiadmin:

Purification of GST-fusion proteins

2025-06-05T12:56:45Z

ESO wikiadmin:

{{Protokoll-Mix|titel=Materials}}

== Procedure ==
The frozen bacteria pellets are thawed in 5 ml T-buffer on ice and resuspended. Afterwards the samples are incubated with 1 ml lysozyme for 30 min. The cells are broken through the addition of 20 ml lysis buffer and the treatment of ultrasonic sound (impulse 50%, 20sec, 3-5x) on ice water.

300 µl sepharose-solution is added to the suspension and incubated for 10 min with head-over-head rotation. Afterwards the lysate is centrifuged for 20 min at 16000 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.

Purification of GST-fusion proteins

2025-06-05T12:56:19Z

ESO wikiadmin: Created page with "== Procedure == The frozen bacteria pellets are thawed in 5 ml T-buffer on ice and resuspended. Afterwards the samples are incubated with 1 ml lysozyme for 30 min. The cells are broken through the addition of 20 ml lysis buffer and the treatment of ultrasonic sound (impulse 50%, 20sec, 3-5x) on ice water. 300 µl sepharose-solution is added to the suspension and incubated for 10 min with head-over-head rotation. Afterwards the lysate is centrifuged for 20 min at 16000 r..."

== Procedure ==
The frozen bacteria pellets are thawed in 5 ml T-buffer on ice and resuspended. Afterwards the samples are incubated with 1 ml lysozyme for 30 min. The cells are broken through the addition of 20 ml lysis buffer and the treatment of ultrasonic sound (impulse 50%, 20sec, 3-5x) on ice water.

300 µl sepharose-solution is added to the suspension and incubated for 10 min with head-over-head rotation. Afterwards the lysate is centrifuged for 20 min at 16000 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.

Expression of GST-fusion proteins

2025-06-05T12:53:59Z

ESO wikiadmin:

{{Protokoll-Mix|Inhalt= 
*LB-Medium (10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0,
ad 1l A. bidest)
*Ampicillin (100 µg/ml)
*IPTG (100 mM Isopropylthiogalactosid)
*2xSDS sample buffer 125mM Tris-HCl (pH 6.8), 10% (v/v)-β-
*Mercaptoethanol, 5% (w/v) SDS, 0.1% (w/v) Bromphenolblau, 20% (v/v) glycerin
*T-buffer 100mM Tris (pH 8.0), 5 mM EDTA
*GST-antibody}}

== Procedure ==

=== '''Test expression:''' ===
After cloning, the constructs of the desired GST-fusion protein are transformed in ''E.coli'' BL21. Eight bacterial clones are selected for overexpression analysis. For the overexpression, bacterial clones are incubated in 5 ml antibiotica containing LB- medium for

6 h at 30°C and 220 rpm. After 6 h, 1ml of the culture is centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 µl 2xSDS sample buffer. The remaining culture is induced with 0.1 mM IPTG and incubated overnight at 30°C and 220 rpm. At the next day

0.5 ml of each sample is centrifuged for 1 min at 13000 rpm, the pellet is resuspended in 100 µl 2xSDS sample buffer and heated for 10 min at 90°C. The expression oft he protein is analysed by SDS-Page and Coomassie-staining.

=== Expression for purification: ===
One bacterial clone is incubated in 30 ml antibiotica containing LB-medium overnight at 30°C and 220 rpm. After 12-18h, 300 ml antibiotic containing LB-medium is inoculated with OD600=0,2 of the overnight-culture and incubated at 30°C and 220 rpm. At OD600 = 0.6-0.8 1 ml is removed (sample before induction) and the remaining culture is induced with 0.5 mM IPTG for 3 h at 30°C and 220 rpm. Afterwards 0.5 ml is removed (sample after incuction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 µl 2xSDS sample buffer and heated for 10 min at 90°C. The expression oft the protein is analysed by SDS-Page and Coomassie-staining. The remaining cells are centrifuged for 15 min at 4700 rpm, washed with T-buffer and stored at -80°C.

Expression of GST-fusion proteins

2025-06-05T12:53:43Z

ESO wikiadmin: Created page with "{{Protokoll-Mix|Inhalt=* *LB-Medium (10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0, ad 1l A. bidest) *Ampicillin (100 µg/ml) *IPTG (100 mM Isopropylthiogalactosid) *2xSDS sample buffer 125mM Tris-HCl (pH 6.8), 10% (v/v)-β- *Mercaptoethanol, 5% (w/v) SDS, 0.1% (w/v) Bromphenolblau, 20% (v/v) glycerin *T-buffer 100mM Tris (pH 8.0), 5 mM EDTA *GST-antibody}} == Procedure == === '''Test expression:''' === After cloning, the constructs of the desired GST-fusion pr..."

{{Protokoll-Mix|Inhalt=*
*LB-Medium (10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0,
ad 1l A. bidest)
*Ampicillin (100 µg/ml)
*IPTG (100 mM Isopropylthiogalactosid)
*2xSDS sample buffer 125mM Tris-HCl (pH 6.8), 10% (v/v)-β-
*Mercaptoethanol, 5% (w/v) SDS, 0.1% (w/v) Bromphenolblau, 20% (v/v) glycerin
*T-buffer 100mM Tris (pH 8.0), 5 mM EDTA
*GST-antibody}}

== Procedure ==

=== '''Test expression:''' ===
After cloning, the constructs of the desired GST-fusion protein are transformed in ''E.coli'' BL21. Eight bacterial clones are selected for overexpression analysis. For the overexpression, bacterial clones are incubated in 5 ml antibiotica containing LB- medium for

6 h at 30°C and 220 rpm. After 6 h, 1ml of the culture is centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 µl 2xSDS sample buffer. The remaining culture is induced with 0.1 mM IPTG and incubated overnight at 30°C and 220 rpm. At the next day

0.5 ml of each sample is centrifuged for 1 min at 13000 rpm, the pellet is resuspended in 100 µl 2xSDS sample buffer and heated for 10 min at 90°C. The expression oft he protein is analysed by SDS-Page and Coomassie-staining.

=== Expression for purification: ===
One bacterial clone is incubated in 30 ml antibiotica containing LB-medium overnight at 30°C and 220 rpm. After 12-18h, 300 ml antibiotic containing LB-medium is inoculated with OD600=0,2 of the overnight-culture and incubated at 30°C and 220 rpm. At OD600 = 0.6-0.8 1 ml is removed (sample before induction) and the remaining culture is induced with 0.5 mM IPTG for 3 h at 30°C and 220 rpm. Afterwards 0.5 ml is removed (sample after incuction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 µl 2xSDS sample buffer and heated for 10 min at 90°C. The expression oft the protein is analysed by SDS-Page and Coomassie-staining. The remaining cells are centrifuged for 15 min at 4700 rpm, washed with T-buffer and stored at -80°C.

Bacterial Pulldown with soluble receptors

2025-06-05T12:50:50Z

ESO wikiadmin: Created page with "{{Protokoll-Mix|titel=Materials|Inhalt=PBS (1x) – 24g NaCl, 0.6g KCl, 3.45g Na2HPO4*7H2O, 0.6g KH2PO4, pH 7.4, ad 1l A.bidest Cell culture supernatants (e.g. OptiMEM) harvested from 293cells transiently transfected with constructs encoding soluble CEACAM- ectodomains fused to GFP Polyclonal rabbit anti-GFP antibody (AG Hauck) 2xSDS sample buffer 125mM Tris-HCl (pH 6.8), 10% (v/v) β-Mercaptoethanol, 5% (w/v) SDS, 0.1% (w/v) Bromphenolblau, 20% (v/v) Glycerin}}..."

{{Protokoll-Mix|titel=Materials|Inhalt=PBS (1x) – 24g NaCl, 0.6g KCl, 3.45g Na2HPO4*7H2O, 0.6g KH2PO4, pH 7.4, ad 1l A.bidest Cell culture supernatants (e.g. OptiMEM) harvested from 293cells transiently transfected with constructs encoding soluble CEACAM- ectodomains fused to GFP Polyclonal rabbit anti-GFP antibody (AG Hauck) 2xSDS sample buffer 125mM Tris-HCl (pH 6.8), 10% (v/v) β-Mercaptoethanol, 5% (w/v) SDS, 0.1% (w/v) Bromphenolblau, 20% (v/v) Glycerin}}

== Procedure ==
In case of low-affinity interactions (HopQ, UspA1, OMP P1) cluster the CEACAM- GFP protein before the Pulldown:

For each sample 1ml of soluble receptor supernatant is incubated with 1µl polyclonal GFP antibody rotating, over night at 4°C.

In case of high-affinity interactions (Opa proteins): there is no need to cluster the CEACAM-GFP protein before Pulldown. Supernatants can be used straight with the bacteria.

Bacteria are grown overnight for 18 h on LB agar plates. Bacteria are lifted from plates with a sterile cotton-tip, suspended in PBS and their OD600 (Ecoli, Hemophilus, Moraxella) or OD550 (Neisseria) is meassured. Bacteria (OD600 0.2) are suspended in cell culture supernatant containing the indicated receptor protein. Bacteria are incubated with equal amounts of the soluble receptor domains for 1 h at 20 °C with head-over-head rotation. After incubation, bacteria are centrifuged at 7000rpm, 5min and subsequently washed twice with PBS and either boiled in SDS sample buffer prior to SDS-PAGE and Western blotting or taken up in PBS and analysed by flow cytometry.

Opsonizing bacteria with Fc fusion proteins

2025-06-05T12:48:33Z

ESO wikiadmin: Created page with "Bacteria are stained with CFSE and resuspended in PBS. 0.3 bacteria (OD 550) are incubated with either 0.05 mg of purified fusion proteins of 1ml of the unpurified fusion protein for 1 h under rotation at 4°C. As a positive control bacteria are incubated with 500 µl of anti-gonococcal sera (heat-inactivated for 1 h at 56°C) and 500 µl PBS. As a negative control bacteria are incubated with 1 ml of PBS. After the incubation the bacteria are washed and resuspended in 1..."

Bacteria are stained with CFSE and resuspended in PBS. 0.3 bacteria (OD 550) are incubated with either 0.05 mg of purified fusion proteins of 1ml of the unpurified fusion protein for 1 h under rotation at 4°C. As a positive control bacteria are incubated with 500 µl of anti-gonococcal sera (heat-inactivated for 1 h at 56°C) and 500 µl PBS. As a negative control bacteria are incubated with 1 ml of PBS. After the incubation the bacteria are washed and resuspended in 1 ml PBS + 2 µl anti-FC antibody for 30 min at 4°C under rotation. Before infection, bacteria are washed once more with PBS.

Lab Etiquette (ENG)

2025-06-05T12:20:20Z

ESO wikiadmin:

'''Diese Seite ist auch auf [[Lab Etiquette|Deutsch]] 🇩🇪 verfügbar.'''<div style="border: 1px solid #ccc; background: #f9f9f9; padding: 1em; margin-bottom: 1em;">
'''Welcome to the lab – think together, act together!'''

A well-functioning lab doesn’t rely solely on rules but on mutual awareness and shared responsibility. Especially for newcomers, it’s better to ask one question too many than to do the wrong thing silently. If you notice that something is running out – let people know. If you change something, inform others. That’s how we keep things smooth, safe, and effective.
</div>

= Laboratory Manual - Cell Biology Research Group =

== Laboratory Access ==
The ML laboratory building is open on weekdays from 6:30 AM to 8:30 PM. Access to laboratory rooms is obtained using RFID transponders, which also allow entry to the laboratory building outside the above opening hours. Master's students and doctoral candidates receive a personal transponder through key distribution (form available from Petra Schnurr) and must return the transponder after completing their time as employees. For bachelor's students and research assistants, a loan transponder is available in exceptional cases after training for necessary work outside opening hours. The loan transponder is located in the key cabinet at the general computer workstation. Removal of the loan transponder must be noted in the list with date and initials.

== Working Hours ==
Non-scientific staff and research assistants are required to record their working time. The online website of the personnel administration is available for this purpose:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

Research assistants use timesheets for time recording (available from Petra Schnurr).

Scientific staff should be present during core working hours 9:00 AM – 12:00 PM and 2:00 PM – 4:00 PM. Absence must be discussed with Christof and noted in the wall calendar in the entrance area of the office row.

== Illness ==
In case of illness, Christof and the secretariat should be notified as soon as possible by telephone and/or email so that coverage, etc. can be organized. For prolonged illness, a medical certificate/medical attestation must be sent to petra.schnurr@uni-konstanz.de on the third day of absence.

== Vacation ==
Employees should apply for vacation as early as possible (at least 1 week in advance) through the online time recording system:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

The possibility for long-term vacation (> 2 weeks) is project-dependent and should be discussed with Christof at least 2 months in advance. During the second half of the winter semester (early January – end of February), vacation during the VTK is only possible in exceptional cases.

Before taking vacation, the culture/storage of important clones/cell lines/samples must always be ensured and coverage for lab duties must be organized. Bachelor's/Master's students should discuss multi-day absences with their supervisor and Christof.

== Seminars/Project Meetings ==
Monday mornings (8:45 AM) there is a general lab meeting. This is followed by project presentations by individual staff members, which are announced on the notice board in the entrance area.

Wednesday afternoons from 3:15 PM – approximately 5:00 PM our journal club takes place. The publication being discussed is loaded onto the file server or sent as a PDF file to everyone the week before. Every two weeks in this seminar, we discuss current new data from all staff members.

Participation in both seminars is mandatory; the seminar schedule is usually set three to six months in advance.

Tuesday afternoons and Thursday lunchtimes during the lecture period, the seminars of the graduate school KoRS-CB as well as the departmental seminar of the Biology Department take place. Speakers/topics are announced by posting. Participation is optional, but when possible, one should not miss interesting presentations.

Specific project meetings take place by arrangement with Christof. Lab books should be brought to project meetings as a basis for discussion.

== Name Abbreviations/Labels ==
Each employee uses a distinctive name abbreviation with which all samples, solutions, etc. must be labeled. Name abbreviations are assigned by the secretariat and are stored in a list on the file server:

Fileserver_LSH/Templates/Lab-Directories/Name-Abbreviations_AG Hauck_2025.doc

== Lab Book ==
Each employee maintains a lab book, which is issued by the secretariat and in which all work is documented in chronological order. Templates for documenting various experiments are stored as PDF files on the file server:

Fileserver_LSH/Templates/Lab-Documentation/

* Gentamicin Assay: "Steffi"
* PCR: "PCR-based-LIC-cloning-protocol-CRH.pdf"
* Cell transfection: "HEKcell-transfection-Protocol-JWK.pdf"
* In vitro Kinase Assay: "Erik"
* Immunoprecipitation: "Marleen"
* Protein expression and purification: "Sarah"
* Lentivirus production: "Marlene"
* Bacterial growth curve: "Ann-Kathrin"

The pages of the lab book are numbered consecutively and a table of contents is created on the first two pages.

The lab book belongs to the Cell Biology Department and remains the property of the department after departure. No pages may be removed from the lab book. Original printouts, gel copies, X-ray films, etc. are labeled and attached to the lab book by being stapled or glued to the corresponding experiments. Electronic original data is stored on the file server in a separate user directory (see Data Archiving chapter).

For most experiments, there are already lab protocols that are described in detail in our VTK script and can be referenced in the lab book. The VTK script can be found on the file server:

''Fileserver_LSH/Templates/Lab-Documentation/Script-Advanced-Course-2025.doc''

The reagents and solutions used are also described either in the recipe file: ''Fileserver_LSH/Recipes/Recipes-2025.doc'' or can be found in the chemical list:

''Fileserver_LSH/Chemical-List/Chemical-List 2025 Group Hauck.xls''

'''Only when deviating from existing protocols are special notes in the lab book necessary.'''

For microscopic work, flow cytometric analyses, or similar work with results in electronic form, a sample protocol is prepared (see Fileserver_LSH/Templates/Lab-Documentation/Protocol-Microscope-FACS.doc") and attached to the experiment.

During project meetings, the lab book serves as the basis for discussing results and should therefore definitely be brought along.

== Data Archiving ==
Standard experiments should be performed according to the lab protocols listed above; deviations should be recorded in the lab book. The raw data obtained during experiments (e.g., microscopy images, gel images, FACS data, VarioScan data) as well as derived figures created from them (graphics, processed microscopy images, etc.) should be stored on the data file server "Fileserver_LSH_Data/00_User/" in a separate user folder. Only there is automatic (daily) backup performed, so no data can be lost.

Each electronic data file contains the date, name abbreviation, and experiment in the name (The raw data files are named according to the following scheme):

'''Year-Month-Day_Name-Abbreviation_Experiment / year-month-day_initials_experiment'''

e.g. /e.g.: 2025-01-01_CRH_Adhesion assay

* the digital version/raw files should be on the file server with the date that can also be found in the lab book
* a printed version of the evaluated data should be attached in the lab book

For later use in publications it is important that we all stick to the same folder structure and naming of files. An example of such a user folder is given on the Fileserver and depicted on the next page:

Fileserver_LSH_Data/00_User/xUE_Universal_Example/

'''Data'''

* ''Constructs''
** SuperSH2 agarose gels, sequencing results, plasmid map, etc.
* ''Western Blots''
* ''Immunofluorescence staining''
* ''Flow Cytometry''
* ''ELISA/SEAP-Assays''
* ''...''

'''Literature'''

'''Presentations'''

'''Thesis:''' for your thesis: for '''every''' figure in your thesis create a subfolder named with the Figure-Number and include in each subfolder both: the final figure as pdf- and illustrator-file and the '''used raw data''' (subdivided in panels A, B, etc.). The name of the raw data file should remain the same as it is found in your DATA-folder and lab book)
[[File:Labetiquette fileserver.jpg|thumb|center|696x696px|'''Fig. 1:''' Example of the user folder structure on the Fileserver_LSH_Data of user UE_Universal_Example.]]

== Laboratory Workspace ==
In the laboratories, there are general laboratory workspaces (e.g., in the two cell culture labs, in the infection room, or the area with protein gel electrophoresis) as well as private workspaces that are assigned. Work at private workspaces of others should only occur after consultation. Everyone is responsible for keeping their workspace, associated shelves, and drawers clean. The work surface is wiped down and disinfected at least once per week; drawers/shelves should be dusted regularly and kept tidy.

Additionally, everyone has a storage space in a refrigerator at 4°C, in a freezer/compartment at -20°C, and can store samples in labeled boxes in the -80°C freezer in room ML. Plastic boxes with lids are available for storage; open stands (especially for Eppendorf tubes) should only be used for short-term storage (1 day). All samples (Eppendorf tubes, agar plates, etc.) must be labeled with abbreviation, content description, and date. All storage locations should be organized/updated regularly (at least once per month). For storage in the -80°C freezer, a data sheet must be filed in the -80°C folder for each box. A template for the data sheet can be found on the file server:

''Fileserver_LSH/Templates/Lab-Documentation/-80C Box-Template.doc''

Please reduce opening of deep freezers to a minimum (otherwise icing and malfunction). Therefore, think before opening about what should be removed and where it can be found (see data sheet!!!). General laboratory workspaces should be cleaned after each use (e.g., sterile bench, protein gel electrophoresis, fume hood, scales, gel documentation) and all used solutions, equipment, etc. must be put away.

== Lab Jobs ==
Each employee receives a lab job that they perform for the general community. The lab job can include maintaining a general collection, responsibility for a device, or supervision and organization of a laboratory. In addition, the preparation of solutions/buffers etc. for the general community is handled through lab jobs. Those responsible are listed on the notice board at the entrance. Room supervisors also organize regular maintenance/cleaning schedules for general equipment such as sterile benches, incubators, etc.

== Closing Service ==
The evening closing service, which ensures that equipment such as sterile benches, vacuum pumps, gel documentation, or the propane gas line are turned off, rotates weekly among scientific staff and master's students. A list for the evening control round of the closing service hangs on the notice board at the entrance.

== Computer Use ==
There is a computer for general use in the seminar room. Please always enable use for official purposes. Private PCs/notebooks can be connected to the network. A current virus scanner and activation of a firewall are required. Access to the department's network storage in the computing center (Fileserver_LSH and Fileserver_LSH_Data) is set up by Christof. Various software programs are available on Fileserver_LSH_Data/02_SOFTWARE, including Adobe Photoshop CS4, Adobe Acrobat Pro9, Adobe Illustrator CS4, MS Office 2016, Clone Manager 9, Endnote X9, SigmaStat 4.0, Leica LAS, Biorad QuantityOne, VarioScan SkanIt. To ensure smooth data exchange within the lab, the above-mentioned versions of these programs must be used for all official purposes, or the data must be saved in a format compatible with these program versions.

== Everyone's Cooperation is Required ==
Each employee can contribute with little effort to minimizing additional work in the laboratory for everyone. Thus, everyone should keep consumption/waste/cleanup effort as low as possible through forward-thinking planning and also help with waste disposal. Please do not fill autoclave bags until "overflowing," but close them in time, deposit them in the tray, and put a new autoclave bag in the corresponding waste container.

Everyone is also responsible for filling boxes with pipette tips and refilling Eppendorf tubes. Autoclave materials are collected in the plastic box and autoclaved by Claudia/Susanne for everyone.

Proper behavior also includes conduct and cleanliness in the tea kitchen and social room. If I have never loaded or unloaded the dishwasher or refilled the coffee machine during the course of one month, then I have very likely used clean dishes at the expense of others.

== Chemicals/Solutions ==
Many solutions are prepared as lab jobs and either kept accessible to everyone as stock solutions in designated places or provided as aliquoted solutions, where one takes an aliquot to their own workspace as needed and stores it there for further use. Additional solutions for personal use should be prepared yourself (please estimate the required amounts in advance and do not prepare liter-wise solutions when you only need 10 ml for two experiments in total) and correctly labeled (abbreviation, date, content: designation and concentration; hazard symbol) and stored at your own workspace. Use solutions and aliquots at colleagues' workspaces ONLY after consultation. Stock solutions, aliquoted solutions, and the respective responsible persons are listed on the "Lab Jobs" bulletin board. For preparing solutions, there is a recipe book available as a file on the file server:

Fileserver_LSH/Recipes/Recipes-2025.doc

== General Collections ==
The department maintains various collections that represent an important resource for everyone and must therefore be handled with particular care and responsibility.

'''Strain Collection:''' includes all bacterial strains that are filed by numbers. Storage at -70°C in chest on level 5 as well as in room ML6:

* non-pathogenic bacteria (= simultaneously repository of all produced plasmids)
* pathogenic bacteria and mutants (key available from Christof)

The strain collection is also available as an MS Access file. Each newly produced or externally obtained bacterial strain receives a new strain number and is frozen in the strain collection as well as electronically recorded. Number distribution is handled by Christof. Only Susanne and Christof have access to the strain collection.

'''Plasmid Collection:''' includes all isolated plasmids from non-pathogenic bacterial strains of the strain collection. Numbering is identical to the strain collection (storage in general refrigerator at 4°C)

'''Cell Collection:''' includes stocks of all cell lines and primary cells. Stored in liquid nitrogen in the media kitchen. The cell collection is managed by Susanne.

'''Virus Collection:''' includes all lentiviral, adenoviral, AAV particles, and bacteriophages. Stored at -70°C. Access file!

'''Oligo Collection:''' includes all oligos ordered for cloning, PCRs, etc. Oligos are stored as 10 mM stock solution in TE buffer at -20°C in the general collection, not in private freezers. Ordering new oligos and filing information is handled by Jan Kuiper.

'''Antibody Collection:''' Collection of all primary antibodies (commercial or self-made) and secondary antibodies (enzyme- or fluorescence-conjugated). Data sheets are collected in the "Antibody" folder. Claudia Hentschel organizes the antibody collection.

'''Protein Collection:''' All recombinant, purified proteins are recorded here.

'''Inhibitor Collection:''' Information on all pharmacological inhibitors and the concentration, solvent, and storage location of stock solutions are filed here. _______________ organizes the inhibitor collection.

'''Enzyme Collection:''' includes all commercial and self-made (DpnI) restriction enzymes stored at -20°C. Removal of enzymes from stock containers only with FILTER TIPS!

Enzymes may only be removed from the freezer for the shortest possible time (1-2 minutes) (i.e., directly before use) and are stored during this short time in a paraffin block cooled to -20°C.

''Folders with documentation for all collections can be found on the shelves at the general workspace in the office area.''

== Competent Cells ==
Aliquots of competent bacteria are stored at -80°C. 200 μl are frozen per aliquot, sufficient for 2 transformations. Various strains are available for different purposes. The standard cloning strain is NovaBlue. When removing, proceed as quickly as possible; warming of aliquots leads to reduced competence; do not remove more aliquots than actually needed.

== Enzymes ==
Polymerases, restriction enzymes, etc. are stored at -20°C in boxes or on paraffin blocks together with the corresponding buffers. These enzymes are used jointly by everyone. Therefore, handle enzyme stock solutions particularly cleanly and avoid contamination (filter tips!!!) as well as warming.

When removing enzymes, transport the Eppendorf tubes with the stock solution exclusively in a paraffin block cooled to -20°C to the workplace and leave them in it for pipetting. Even with paraffin block, remove the enzyme from the -20°C freezer only briefly (< 2 minutes) and return it immediately after pipetting. When opening stock containers, definitely avoid contact of the inner lid surface with skin!!

== Orders ==
When general chemicals or disposables are running low, give notice IN TIME (i.e., before using up the last package or pipetting the last microliter): either write on the board in the lab or tell the orderers. Discuss project-specific chemicals/antibodies etc. or lab utensils/equipment with Christof before ordering.

For oligo orders through Jan, enter the oligo information in the order list on the file server: Fileserver_LSH:\Strain-Collection\Primer\01_PrimerOrdering_Primer-Order

Discuss other online orders with Christof.

Order sequencing using pre-paid labels from LGC or GeneWiz. Please no more than 2 sequencing per new construct. Discuss larger sequencing needs with Christof. Deliveries of reagents should be handed over to the orderer. The orderer checks for completeness (using the delivery slip) and confirms with date and signature. The delivery slip and any accompanying invoices are forwarded to Petra Schnurr in the secretariat. When chemicals, antibodies, enzymes, and other reagents are delivered, the data sheets are dated and filed in the corresponding folders (see "General Collections"). The solvent, concentration, and possibly filled aliquots as well as storage location may be noted on the data sheets.

ALL chemicals, reagents, enzymes, etc. are stored in accessible and known locations.

== Laboratory Safety ==
See [[All About Safety|Safety Guidelines]].

Lab Etiquette (ENG)

2025-06-05T12:19:31Z

ESO wikiadmin:

'''Diese Seite ist auch auf [[Lab Etiquette|Deutsch]] 🇩🇪 verfügbar.'''<div style="border: 1px solid #ccc; background: #f9f9f9; padding: 1em; margin-bottom: 1em;">
'''Welcome to the lab – think together, act together!'''

A well-functioning lab doesn’t rely solely on rules but on mutual awareness and shared responsibility. Especially for newcomers, it’s better to ask one question too many than to do the wrong thing silently. If you notice that something is running out – let people know. If you change something, inform others. That’s how we keep things smooth, safe, and effective.
</div>

= Laboratory Manual - Cell Biology Research Group =

== Laboratory Access ==
The ML laboratory building is open on weekdays from 6:30 AM to 8:30 PM. Access to laboratory rooms is obtained using RFID transponders, which also allow entry to the laboratory building outside the above opening hours. Master's students and doctoral candidates receive a personal transponder through key distribution (form available from Petra Schnurr) and must return the transponder after completing their time as employees. For bachelor's students and research assistants, a loan transponder is available in exceptional cases after training for necessary work outside opening hours. The loan transponder is located in the key cabinet at the general computer workstation. Removal of the loan transponder must be noted in the list with date and initials.

== Working Hours ==
Non-scientific staff and research assistants are required to record their working time. The online website of the personnel administration is available for this purpose:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

Research assistants use timesheets for time recording (available from Petra Schnurr).

Scientific staff should be present during core working hours 9:00 AM – 12:00 PM and 2:00 PM – 4:00 PM. Absence must be discussed with Christof and noted in the wall calendar in the entrance area of the office row.

== Illness ==
In case of illness, Christof and the secretariat should be notified as soon as possible by telephone and/or email so that coverage, etc. can be organized. For prolonged illness, a medical certificate/medical attestation must be sent to petra.schnurr@uni-konstanz.de on the third day of absence.

== Vacation ==
Employees should apply for vacation as early as possible (at least 1 week in advance) through the online time recording system:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

The possibility for long-term vacation (> 2 weeks) is project-dependent and should be discussed with Christof at least 2 months in advance. During the second half of the winter semester (early January – end of February), vacation during the VTK is only possible in exceptional cases.

Before taking vacation, the culture/storage of important clones/cell lines/samples must always be ensured and coverage for lab duties must be organized. Bachelor's/Master's students should discuss multi-day absences with their supervisor and Christof.

== Seminars/Project Meetings ==
Monday mornings (8:45 AM) there is a general lab meeting. This is followed by project presentations by individual staff members, which are announced on the notice board in the entrance area.

Wednesday afternoons from 3:15 PM – approximately 5:00 PM our journal club takes place. The publication being discussed is loaded onto the file server or sent as a PDF file to everyone the week before. Every two weeks in this seminar, we discuss current new data from all staff members.

Participation in both seminars is mandatory; the seminar schedule is usually set three to six months in advance.

Tuesday afternoons and Thursday lunchtimes during the lecture period, the seminars of the graduate school KoRS-CB as well as the departmental seminar of the Biology Department take place. Speakers/topics are announced by posting. Participation is optional, but when possible, one should not miss interesting presentations.

Specific project meetings take place by arrangement with Christof. Lab books should be brought to project meetings as a basis for discussion.

== Name Abbreviations/Labels ==
Each employee uses a distinctive name abbreviation with which all samples, solutions, etc. must be labeled. Name abbreviations are assigned by the secretariat and are stored in a list on the file server:

Fileserver_LSH/Templates/Lab-Directories/Name-Abbreviations_AG Hauck_2025.doc

== Lab Book ==
Each employee maintains a lab book, which is issued by the secretariat and in which all work is documented in chronological order. Templates for documenting various experiments are stored as PDF files on the file server:

Fileserver_LSH/Templates/Lab-Documentation/

* Gentamicin Assay: "Steffi"
* PCR: "PCR-based-LIC-cloning-protocol-CRH.pdf"
* Cell transfection: "HEKcell-transfection-Protocol-JWK.pdf"
* In vitro Kinase Assay: "Erik"
* Immunoprecipitation: "Marleen"
* Protein expression and purification: "Sarah"
* Lentivirus production: "Marlene"
* Bacterial growth curve: "Ann-Kathrin"

The pages of the lab book are numbered consecutively and a table of contents is created on the first two pages.

The lab book belongs to the Cell Biology Department and remains the property of the department after departure. No pages may be removed from the lab book. Original printouts, gel copies, X-ray films, etc. are labeled and attached to the lab book by being stapled or glued to the corresponding experiments. Electronic original data is stored on the file server in a separate user directory (see Data Archiving chapter).

For most experiments, there are already lab protocols that are described in detail in our VTK script and can be referenced in the lab book. The VTK script can be found on the file server:

''Fileserver_LSH/Templates/Lab-Documentation/Script-Advanced-Course-2025.doc''

The reagents and solutions used are also described either in the recipe file: ''Fileserver_LSH/Recipes/Recipes-2025.doc'' or can be found in the chemical list:

''Fileserver_LSH/Chemical-List/Chemical-List 2025 Group Hauck.xls''

'''Only when deviating from existing protocols are special notes in the lab book necessary.'''

For microscopic work, flow cytometric analyses, or similar work with results in electronic form, a sample protocol is prepared (see Fileserver_LSH/Templates/Lab-Documentation/Protocol-Microscope-FACS.doc") and attached to the experiment.

During project meetings, the lab book serves as the basis for discussing results and should therefore definitely be brought along.

== Data Archiving ==
Standard experiments should be performed according to the lab protocols listed above; deviations should be recorded in the lab book. The raw data obtained during experiments (e.g., microscopy images, gel images, FACS data, VarioScan data) as well as derived figures created from them (graphics, processed microscopy images, etc.) should be stored on the data file server "Fileserver_LSH_Data/00_User/" in a separate user folder. Only there is automatic (daily) backup performed, so no data can be lost.

Each electronic data file contains the date, name abbreviation, and experiment in the name (The raw data files are named according to the following scheme):

'''Year-Month-Day_Name-Abbreviation_Experiment / year-month-day_initials_experiment'''

e.g. /e.g.: 2025-01-01_CRH_Adhesion assay

* the digital version/raw files should be on the file server with the date that can also be found in the lab book
* a printed version of the evaluated data should be attached in the lab book

For later use in publications it is important that we all stick to the same folder structure and naming of files. An example of such a user folder is given on the Fileserver and depicted on the next page:

Fileserver_LSH_Data/00_User/xUE_Universal_Example/

'''Data'''

* ''Constructs''
** SuperSH2 agarose gels, sequencing results, plasmid map, etc.
* ''Western Blots''
* ''Immunofluorescence staining''
* ''Flow Cytometry''
* ''ELISA/SEAP-Assays''
* ''...''

'''Literature'''

'''Presentations'''

'''Thesis:''' for your thesis: for '''every''' figure in your thesis create a subfolder named with the Figure-Number and include in each subfolder both: the final figure as pdf- and illustrator-file and the '''used raw data''' (subdivided in panels A, B, etc.). The name of the raw data file should remain the same as it is found in your DATA-folder and lab book)
[[File:Labetiquette fileserver.jpg|thumb]]

== Laboratory Workspace ==
In the laboratories, there are general laboratory workspaces (e.g., in the two cell culture labs, in the infection room, or the area with protein gel electrophoresis) as well as private workspaces that are assigned. Work at private workspaces of others should only occur after consultation. Everyone is responsible for keeping their workspace, associated shelves, and drawers clean. The work surface is wiped down and disinfected at least once per week; drawers/shelves should be dusted regularly and kept tidy.

Additionally, everyone has a storage space in a refrigerator at 4°C, in a freezer/compartment at -20°C, and can store samples in labeled boxes in the -80°C freezer in room ML. Plastic boxes with lids are available for storage; open stands (especially for Eppendorf tubes) should only be used for short-term storage (1 day). All samples (Eppendorf tubes, agar plates, etc.) must be labeled with abbreviation, content description, and date. All storage locations should be organized/updated regularly (at least once per month). For storage in the -80°C freezer, a data sheet must be filed in the -80°C folder for each box. A template for the data sheet can be found on the file server:

''Fileserver_LSH/Templates/Lab-Documentation/-80C Box-Template.doc''

Please reduce opening of deep freezers to a minimum (otherwise icing and malfunction). Therefore, think before opening about what should be removed and where it can be found (see data sheet!!!). General laboratory workspaces should be cleaned after each use (e.g., sterile bench, protein gel electrophoresis, fume hood, scales, gel documentation) and all used solutions, equipment, etc. must be put away.

== Lab Jobs ==
Each employee receives a lab job that they perform for the general community. The lab job can include maintaining a general collection, responsibility for a device, or supervision and organization of a laboratory. In addition, the preparation of solutions/buffers etc. for the general community is handled through lab jobs. Those responsible are listed on the notice board at the entrance. Room supervisors also organize regular maintenance/cleaning schedules for general equipment such as sterile benches, incubators, etc.

== Closing Service ==
The evening closing service, which ensures that equipment such as sterile benches, vacuum pumps, gel documentation, or the propane gas line are turned off, rotates weekly among scientific staff and master's students. A list for the evening control round of the closing service hangs on the notice board at the entrance.

== Computer Use ==
There is a computer for general use in the seminar room. Please always enable use for official purposes. Private PCs/notebooks can be connected to the network. A current virus scanner and activation of a firewall are required. Access to the department's network storage in the computing center (Fileserver_LSH and Fileserver_LSH_Data) is set up by Christof. Various software programs are available on Fileserver_LSH_Data/02_SOFTWARE, including Adobe Photoshop CS4, Adobe Acrobat Pro9, Adobe Illustrator CS4, MS Office 2016, Clone Manager 9, Endnote X9, SigmaStat 4.0, Leica LAS, Biorad QuantityOne, VarioScan SkanIt. To ensure smooth data exchange within the lab, the above-mentioned versions of these programs must be used for all official purposes, or the data must be saved in a format compatible with these program versions.

== Everyone's Cooperation is Required ==
Each employee can contribute with little effort to minimizing additional work in the laboratory for everyone. Thus, everyone should keep consumption/waste/cleanup effort as low as possible through forward-thinking planning and also help with waste disposal. Please do not fill autoclave bags until "overflowing," but close them in time, deposit them in the tray, and put a new autoclave bag in the corresponding waste container.

Everyone is also responsible for filling boxes with pipette tips and refilling Eppendorf tubes. Autoclave materials are collected in the plastic box and autoclaved by Claudia/Susanne for everyone.

Proper behavior also includes conduct and cleanliness in the tea kitchen and social room. If I have never loaded or unloaded the dishwasher or refilled the coffee machine during the course of one month, then I have very likely used clean dishes at the expense of others.

== Chemicals/Solutions ==
Many solutions are prepared as lab jobs and either kept accessible to everyone as stock solutions in designated places or provided as aliquoted solutions, where one takes an aliquot to their own workspace as needed and stores it there for further use. Additional solutions for personal use should be prepared yourself (please estimate the required amounts in advance and do not prepare liter-wise solutions when you only need 10 ml for two experiments in total) and correctly labeled (abbreviation, date, content: designation and concentration; hazard symbol) and stored at your own workspace. Use solutions and aliquots at colleagues' workspaces ONLY after consultation. Stock solutions, aliquoted solutions, and the respective responsible persons are listed on the "Lab Jobs" bulletin board. For preparing solutions, there is a recipe book available as a file on the file server:

Fileserver_LSH/Recipes/Recipes-2025.doc

== General Collections ==
The department maintains various collections that represent an important resource for everyone and must therefore be handled with particular care and responsibility.

'''Strain Collection:''' includes all bacterial strains that are filed by numbers. Storage at -70°C in chest on level 5 as well as in room ML6:

* non-pathogenic bacteria (= simultaneously repository of all produced plasmids)
* pathogenic bacteria and mutants (key available from Christof)

The strain collection is also available as an MS Access file. Each newly produced or externally obtained bacterial strain receives a new strain number and is frozen in the strain collection as well as electronically recorded. Number distribution is handled by Christof. Only Susanne and Christof have access to the strain collection.

'''Plasmid Collection:''' includes all isolated plasmids from non-pathogenic bacterial strains of the strain collection. Numbering is identical to the strain collection (storage in general refrigerator at 4°C)

'''Cell Collection:''' includes stocks of all cell lines and primary cells. Stored in liquid nitrogen in the media kitchen. The cell collection is managed by Susanne.

'''Virus Collection:''' includes all lentiviral, adenoviral, AAV particles, and bacteriophages. Stored at -70°C. Access file!

'''Oligo Collection:''' includes all oligos ordered for cloning, PCRs, etc. Oligos are stored as 10 mM stock solution in TE buffer at -20°C in the general collection, not in private freezers. Ordering new oligos and filing information is handled by Jan Kuiper.

'''Antibody Collection:''' Collection of all primary antibodies (commercial or self-made) and secondary antibodies (enzyme- or fluorescence-conjugated). Data sheets are collected in the "Antibody" folder. Claudia Hentschel organizes the antibody collection.

'''Protein Collection:''' All recombinant, purified proteins are recorded here.

'''Inhibitor Collection:''' Information on all pharmacological inhibitors and the concentration, solvent, and storage location of stock solutions are filed here. _______________ organizes the inhibitor collection.

'''Enzyme Collection:''' includes all commercial and self-made (DpnI) restriction enzymes stored at -20°C. Removal of enzymes from stock containers only with FILTER TIPS!

Enzymes may only be removed from the freezer for the shortest possible time (1-2 minutes) (i.e., directly before use) and are stored during this short time in a paraffin block cooled to -20°C.

''Folders with documentation for all collections can be found on the shelves at the general workspace in the office area.''

== Competent Cells ==
Aliquots of competent bacteria are stored at -80°C. 200 μl are frozen per aliquot, sufficient for 2 transformations. Various strains are available for different purposes. The standard cloning strain is NovaBlue. When removing, proceed as quickly as possible; warming of aliquots leads to reduced competence; do not remove more aliquots than actually needed.

== Enzymes ==
Polymerases, restriction enzymes, etc. are stored at -20°C in boxes or on paraffin blocks together with the corresponding buffers. These enzymes are used jointly by everyone. Therefore, handle enzyme stock solutions particularly cleanly and avoid contamination (filter tips!!!) as well as warming.

When removing enzymes, transport the Eppendorf tubes with the stock solution exclusively in a paraffin block cooled to -20°C to the workplace and leave them in it for pipetting. Even with paraffin block, remove the enzyme from the -20°C freezer only briefly (< 2 minutes) and return it immediately after pipetting. When opening stock containers, definitely avoid contact of the inner lid surface with skin!!

== Orders ==
When general chemicals or disposables are running low, give notice IN TIME (i.e., before using up the last package or pipetting the last microliter): either write on the board in the lab or tell the orderers. Discuss project-specific chemicals/antibodies etc. or lab utensils/equipment with Christof before ordering.

For oligo orders through Jan, enter the oligo information in the order list on the file server: Fileserver_LSH:\Strain-Collection\Primer\01_PrimerOrdering_Primer-Order

Discuss other online orders with Christof.

Order sequencing using pre-paid labels from LGC or GeneWiz. Please no more than 2 sequencing per new construct. Discuss larger sequencing needs with Christof. Deliveries of reagents should be handed over to the orderer. The orderer checks for completeness (using the delivery slip) and confirms with date and signature. The delivery slip and any accompanying invoices are forwarded to Petra Schnurr in the secretariat. When chemicals, antibodies, enzymes, and other reagents are delivered, the data sheets are dated and filed in the corresponding folders (see "General Collections"). The solvent, concentration, and possibly filled aliquots as well as storage location may be noted on the data sheets.

ALL chemicals, reagents, enzymes, etc. are stored in accessible and known locations.

== Laboratory Safety ==
See [[All About Safety|Safety Guidelines]].

Lab Etiquette

2025-06-05T12:17:56Z

ESO wikiadmin:

'''Diese Seite ist auch auf [[Lab Etiquette (ENG)|Englisch 🇬🇧]] verfügbar.'''

== '''Laborzugang''' ==
Das Laborgebäude ML ist zwischen 6:30 und 20:30 Uhr an Werktagen geöffnet. Zugang zu den Laborräumen erhält man mittels RFID-Transponder, der es auch erlaubt, das Laborgebäude außerhalb der oben stehenden Öffnungszeiten zu betreten. Masterstudenten und Doktoranden erhalten einen persönlichen Transponder über die Schlüsselausgabe (Formular bei Petra Schnurr) und müssen den Transponder nach Beendigung ihrer Zeit als Mitarbeiter wieder abgeben. Für Bachlorstudenten und Hiwis gibt es nach Einarbeitung in Ausnahmefällen einen Leihtransponder für notwendige Arbeiten außerhalb der Öffnungszeiten. Der Leihtransponder befindet sich im Schlüsselkasten am allg. Computer-Arbeitsplatz. Die Entnahme des Leihtransponders ist in der Liste mit Datum und Kürzel zu vermerken.

== '''Arbeitszeiten''' ==
Nicht-wissenschaftliche Mitarbeiter und Hiwis sind verpflichtet, die Arbeitszeit zu erfassen. Hierzu steht den Mitarbeitern die Online-Webseite der Personalverwaltung zur Verfügung:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

Hiwis nutzen zur Zeiterfassung den Stundenzettel (erhältlich bei Petra Schnurr)

Wissenschaftliche Mitarbeiter sollen zu den Kernarbeitszeiten 9.00 – 12.00 und 14.00 – 16.00 anwesend sein. Eine Abwesenheit ist mit Christof abzusprechen und im Wandkalender im Eingangsbereich der Bürozeile zu vermerken.

== '''Krankheit''' ==
Im Krankheitsfall soll so bald wie möglich Christof und das Sekretariat per Telefon und/oder E-Mail benachrichtigt werden, damit Vertretung, etc. organisiert werden kann. Bei längerer Krankheit muss am dritten Tag der Abwesenheit eine ärztliche Krankmeldung/ein ärztliches Attest an petra.schnurr@uni-konstanz.de geschickt werden.

== '''Urlaub''' ==
Mitarbeiter beantragen möglichst frühzeitig (mindestens 1 Woche vorher) ihren Urlaub über das online Zeiterfassungssystem:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

Die Möglichkeit für längerfristigen Urlaub (> 2 Wochen) ist projektabhängig und soll mindestens 2 Monate vorher mit Christof abgesprochen werden. In der 2.Hälfte des Wintersemesters (Anfang Januar – Ende Februar) ist während des VTK Urlaub nur in Ausnahmefällen möglich.

Vor Urlaubsantritt muss immer die Kultur/Lagerung von wichtigen Klonen/Zellinien/Proben sichergestellt und eine Vertretung für den Labjob organisiert sein. Bachelor-/Masterstudenten sprechen eine mehrtägige Abwesenheit mit Ihrem/Ihrer BetreuerIn und Christof ab.

== '''Seminare/Projektbesprechungen''' ==
Montags vormittags (8.45 Uhr) ist eine allg. Laborbesprechung. Im Anschluß daran eine Porjektvorstellung einzelner Mitarbeiter, die am Notice-Board im Eingangsbereich angekündigt wird.

Mittwochs am Nachmittag von 15.15 Uhr – ca. 17.00 Uhr findet unser Journal-Club statt. Die jeweils behandelte Publikation wird in der Woche zuvor auf den Fileserver geladen bzw. als pdf-file an alle verschickt. Im 14-tägigen Turnus besprechen wir in diesem Seminar aktuelle neue Daten aller Mitarbeiter.

Die Teilnahme an diesen beiden Seminaren ist obligatorisch, der Seminarplan wird in der Regel auf ein viertel- bis ein halbes Jahr im voraus festgelegt.

Dienstags nachmittags und donnerstags zur Mittagszeit finden während der Vorlesungszeit die Seminare der Graduiertenschule KoRS-CB sowie das Fachbereichsseminar des FB Biologie statt. Die Vortragenden/Themen werden durch Aushang bekannt gegeben. Die Teilnahme ist fakultativ, aber wenn möglich sollte man sich die interessanten Vorträge nicht entgehen lassen.

Spezifische Projektbesprechungen finden nach Absprache mit Christof statt. Zu den Projektbesprechungen ist das Laborbuch als Gesprächsgrundlage mitzubringen.

== '''Namenskürzel/Beschriftungen''' ==
Jeder Mitarbeiter benutzt ein unverwechselbares Namenskürzel, mit dem alle Proben, Lösungen, etc. beschriftet werden müssen. Die Namenskürzel werden im Sekretariat vergeben und sind in einer Liste auf dem Fileserver abgelegt:

Fileserver_LSH/Vorlagen/Labor-Verzeichnisse/Namenskuerzel_AG Hauck_2025.doc

== '''Laborbuch''' ==
Jeder Mitarbeiter führt ein Laborbuch, das im Sekretariat ausgegeben wird und in dem alle Arbeiten in chronologischer Reihenfolge dokumentiert werden. Muster für die Dokumentation von verschiedenen Experimenten sind als pdf-file auf dem Fileserver abgelegt:

Fileserver_LSH/Vorlagen/Labor-Protokollierung/

* Gentamicin-Assay: „Steffi“
* PCR: „''PCR-based-LIC-cloning-protocol-CRH.pdf''“
* Zelltransfektion: „''HEKcell-transfection-Protocol-JWK.pdf''“
* In vitro Kinase Assay: “Erik”
* [[Immunoprecipitation|Immunopräzipitation]] (MHE)
* Protein expression and purification: “Sarah”
* Lentivirus production: “Marlene”
* Bacterial growth curve: “Ann-Kathrin”

Die Seiten des Laborbuchs werden durchnumeriert und auf den ersten beiden Seiten wird ein Inhaltsverzeichnis angelegt.

Das Laborbuch gehört dem Lehrstuhl Zellbiologie und verbleibt nach dem Ausscheiden im Besitz des Lehrstuhls. Aus dem Laborbuch dürfen keine Seiten entfernt werden. Originalausdrucke, Kopien von Gelen, Röntgenfilme, etc. werden beschriftet und dem Laborbuch beigelegt, indem sie zu den entsprechenden Experimenten geheftet oder geklebt werden. Die elektronischen Originaldaten werden auf dem Fileserver in einem eigenen User-Verzeichnis gespeichert (s. Kapitel Daten Archivierung).

Für die meisten Experimente gibt es bereits Labor-Protokolle, die in unserem VTK-Skript ausführlich beschrieben sind und auf die im Laborbuch verwiesen werden kann. Das VTK-Skript findet sich auf dem Fileserver:

''Fileserver_LSH/Vorlagen/Labor-Protokollierung/Skript-Vertiefungskurs-2025.doc''

Die verwendeten Reagenzien und Lösungen sind ebenfalls entweder in der Rezepte-Datei beschrieben: ''Fileserver_LSH/Rezepte/Rezepte-2025.doc'' oder sind in der Chemikalienliste zu finden:

''Fileserver_LSH/Chemikalienliste/Chemikalienliste 2025 Gruppe Hauck.xls''

Nur bei Abweichen vom existierenden Protokoll sind besondere Vermerke im Laborbuch notwendig.

Bei mikroskopischen Arbeiten, durchflußzytometrischen Analysen oder ähnlichen Arbeiten mit Ergebnissen in elektronischer Form wird ein Probenprotokoll angefertigt (s. Fileserver_LSH/Vorlagen/Labor-Protokollierung/Protokoll-Mikroskop-FACS.doc“) und dem Experiment beigeheftet.

Bei Projektbesprechungen dient das Laborbuch als Grundlage für die Diskussion der Ergebnisse und ist deshalb unbedingt mitzubringen.

== '''Daten-Archivierung''' ==
Standardexperimente sollen anhand der oben angeführten Laborprotokolle durchgeführt werden, Abweichungen sind im Laborbuch festzuhalten. Die im Rahmen der Experimente erhaltenen Rohdaten (z.B. Mikroskopiebilder, Gelbilder, FACS-Daten, VarioScan-Daten) sowie die daraus hergestellten abgeleiteten Abbildungen (Graphiken, berbeiteten Mikroskopiebilder, etc.) sind auf dem Daten-Fileserver „Fileserver_LSH_Data/00_User/„ in einem separaten Nutzerfolder zu speichern. Nur dort erfolgt eine automatische (tägliche) Sicherung, so dass keine Daten verloren gehen können.

Jede elektronische Datendatei trägt im Namen das Datum, das Namenskürzel und das Experiment (The raw data files are named according to the following scheme):

'''Jahr-Monat-Tag_Namenskürzel_Experiment / year-month-day_initials_experiment'''

z.B. /e.g.: 2025-01-01_CRH_Adhesion assay

- the digital version/raw files should be on the file server with the date that can also be found in the lab book

- a printed version of the evaluated data should be attached in the lab book

For later use in publications it is important that we all stick to the same folder structure and naming of files. An example of such a user folder is given on the Fileserver and depicted on the next page:

Fileserver_LSH_Data/00_User/xUE_Universal_Example/

'''Data'''

'' Constructs''

SuperSH2 agarose gels, sequencing results, plasmid map, etc.

'' Western Blots''

'' Immunofluorescence staining''

'' Flow Cytometry''

'' ELISA/SEAP-Assays''

''' .'''

''' .'''

'''Literature'''

'''Presentations'''

'''Thesis:''' for your thesis: for '''every''' figure in your thesis create a subfolder named with the Figure-Number and include in each subfolder both: the final figure as pdf- and illustrator-file and the '''used raw data''' (subdivided in panels A, B, etc.). The name of the raw data file should remain the same as it is found in your DATA-folder and lab book)
[[File:Labetiquette fileserver.jpg|center|thumb|700x700px|'''Fig. 1:''' Example of the user folder structure on the Fileserver_LSH_Data of user UE_Universal_Example.]]

== '''Laborarbeitsplatz''' ==
In den Laboren gibt es allgemeine Laborarbeitsplätze (z.B. in den beiden Zellkulturlaboren, im Infektionsraum oder der Bereich mit Proteingel-Elektrophorese) sowie private Arbeitsplätze, die zugewiesen sind. Arbeiten auf den privaten Arbeitsplätzen anderer soll nur nach Rücksprache erfolgen. Jeder ist für die Sauberhaltung seines Arbeitsplatzes, der dazugehörigen Regale und Schubladen selbst verantwortlich. Die Arbeitsfläche wird mindestens einmal pro Woche abgewischt und desinfiziert, die Schubladen/Regale sind regelmäßig abzustauben und ordentlich zu halten.

Zusätzlich hat jeder einen Lagerplatz in einem Kühlschrank bei 4°C, in einem Gefrierschrank/-fach bei -20°C und kann Proben in beschrifteten Boxen im -80°C-Schrank in Raum ML lagern. Für die Lagerung stehen Kunststoffboxen mit Deckel zur Verfügung, offenen Ständer (speziell für Eppendorfgefäße) sollen nur für Kurzzeit-Aufbewahrung (1 Tag) verwendet werden. Alle Proben (Eppis, Agarplatten, etc.) müssen mit Kürzel, Inhaltsbezeichnung und Datum beschriftet sein. Alle Lagerplätze sollen regelmäßig (mindestens 1x je Monat) aufgeräumt/aktualisiert werden. Bei Lagerung im -80°C Schrank ist für jede Box ein Datenblatt in den -80°C-Ordner abzulegen. Eine Vorlage für das Datenblatt findet sich auf dem Fileserver:

''Fileserver_LSH/Vorlagen/Labor-Protokollierung/'' ''-80C Box-Vorlage.doc''

Das Öffnen von Tiefgefrierschränken bitte auf ein Minimum reduzieren (da sonst Vereisung und Fehlfunktion). Deshalb bereits VOR dem Öffnen überlegen, was entnommen werden soll und wo es zu finden ist (s. Datenblatt!!!). Allgemeine Laborarbeitsplätze sind nach jeder Benutzung zu säubern (z.B. sterile Werkbank, Proteingel-Elektrophorese, Abzug, Waagen, Geldoku) und alle verwendeten Lösungen, Apparaturen, etc. müssen wieder aufgeräumt werden.

== '''Lab-Jobs''' ==
Jeder Mitarbeiter erhält einen Labjob, den er für die Allgemeinheit wahrnimmt. Der Lab-Job kann die Pflege einer allgemeinen Sammlung, die Verantwortung für ein Gerät oder die Aufsicht und Organisation eines Labors beinhalten. Darüber hinaus wird die Herstellung von Lösungen/Puffern etc. für die Allgemeinheit über Lab-Jobs. Die jeweils Verantwortlichen sind auf einer Liste am am Noticeboard am Eingang aufgeführt. Die Raum-Verantwortlichen organisieren auch die regelmäßige Pflege/Putzpläne für allgemeine Geräte wie sterile Werkbänke, Inkubatoren, etc.

== '''Schließdienst''' ==
Der abendliche Schließdienst, der dafür sorgt, dass Geräte wie sterile Werkbänke, Vakkumpumpen, Geldoku oder die Propangasleitung ausgeschaltet sind, wechselt im wöchentlichen Turnus unter den wissenschaftlichen Mitarbeitern und Masterstudenten. Eine Liste für den abendlichen Kontrollgang des Schließdienstes hängt am Noticeboard am Eingang.

== '''Computernutzung''' ==
Im Seminarraum befindet sich ein Computer zur allgemeinen Nutzung. Bitte jederzeit die Nutzung für dienstliche Zwecke ermöglichen. Private PCs/Notebooks können an das Netzwerk angeschlossen werden. Voraussetzung ist ein aktueller Virenscanner und die Aktivierung einer Firewall. Den Zugang zu den Netzwerk-Speichern des Lehrstuhls im Rechenzentrum (Fileserver_LSH und Fileserver_LSH_Data) richtet Christof ein. Auf dem Fileserver_LSH_Data/02_SOFTWARE stehen verschiedene Software-Programme zur Verfügung, darunter Adobe Photoshop CS4, Adobe Acrobat Pro9, Adobe Illustrator CS4, MS Office 2016, Clone Manager 9, Endnote X9, SigmaStat 4.0, Leica LAS, Biorad QuantityOne, VarioScan SkanIt. Um den problemlosen Austausch der Daten innerhalb des Labors zu gewährleisten, müssen die oben angegebenen Versionen dieser Programme für alle dienstlichen Zwecke verwendet werden bzw. die Daten in einer mit diesen Programmversionen kompatiblen Form gespeichert werden.

== '''Die Mitarbeit jedes Einzelnen ist gefragt''' ==
Jeder Mitarbeiter kann durch wenig Aufwand dazu beitragen, dass sich die Zusatzarbeiten im Labor für Alle minimieren. So soll jeder durch vorausschauende Planung den anfallenden Verbrauch/Abfall/Aufräumaufwand möglichst gering halten und auch bei der Entsorgung von Abfällen mithelfen. Bitte Autoklaviersäcke nicht bis zum „Überquellen“ füllen, sondern rechtzeitig verschließen, in der Wanne deponieren und neuen Autoklaviersack in den entsprechenden Müllbehälter geben.

Jeder ist auch selbst für das Befüllen der Boxen mit Pipettenspitzen und das Auffüllen von Eppis verantwortlich. Autoklaviergut wird in der Plastikkiste gesammelt und von Claudia/Susanne für alle autoklaviert.

Zum verträglichen Umgang gehört auch das Verhalten und die sauberkeit in der Teeküche und im Sozialraum. Wenn ich im Verlaufe von 1 Monat nie die Spülmaschine ein- oder ausgeräumt oder die Kaffeemaschine aufgefüllt habe, dann habe ich mich sehr wahrscheinlich auf Kosten von Anderen am sauberen Geschirr bedient.

== '''Chemikalien/Lösungen''' ==
Viele Lösungen werden als Lab-Job angesetzt und entweder als Stocklösung an festgelegten Plätzen für Alle zugänglich aufbewahrt oder als aliquotierte Lösung bereitgestellt, wobei man sich bei Bedarf ein Aliquot an den eigenen Arbeitsplatz mitnimmt und dort für die weitere Verwendung aufbewahrt. Weitere Lösungen für den eigenen Bedarf sind selbst anzusetzen (bitte die benötigten Mengen im Voraus abschätzen und nicht Liter-weise Lösungen ansetzen, wenn man für zwei Experimente insgesamt nur 10 ml braucht) und korrekt beschriftet (Kürzel, Datum, Inhalt: Bezeichnung und Konzentration; Gefahrensymbol) am eigenen Arbeitsplatz aufzubewahren. Lösungen und Aliquots am Arbeitsplatz der Kollegen NUR nach Rücksprache benutzen. Stocklösungen, aliquotierte Lösungen und die jeweiligen Verantwortlichen sind auf der Liste am Schwarzen Brett „Lab-Jobs“ aufgeführt. Für das Ansetzen von Lösungen gibt es ein Rezeptbuch, das als Datei auf dem Fileserver bereit liegt:

Fileserver_LSH//Rezepte/ Rezepte-2025.doc

== '''Allgemeine Sammlungen''' ==
Der Lehrstuhl unterhält verschiedene Sammlungen, die eine wichtige Ressource für Alle darstellen und mit denen deshalb besonders umsichtig und verantwortlich umgegangen werden muss.

Stammsammlung: umfasst alle Bakterienstämme, die nach Nummern abgelegt sind. Lagerung bei -70°C in Truhe auf Ebene 5 sowie in Raum ML6 :

– nichtpathogene Bakterien (=gleichzeitig Repositorium aller hergestellten Plasmide)

– pathogene Bakterien und Mutanten (Schlüssel dafür bei Christof)

Die Stammsammlung ist auch als MS Access-Datei vorhanden. Jeder neu hergestellte oder von extern erhaltene Bakterienstamm erhält eine neue Stammnummer und wird in der Stammsammlung eingefroren sowie elektronisch erfasst. Die Verteilung der Nummern erfolgt über Christof. Zugang zur Stammsammlung haben nur Susanne und Christof.

Plasmidsammlung: umfasst alle isolierten Plasmide aus den nichtpathogenen Bakterienstämmen der Stammsammlung. Die Numerierung ist identisch mit der Stammsammlung (Lagerung im allg. Kühlschrank bei 4°C)

Zellsammlung: umfasst Stocks aller Zellinien und primärer Zellen. Werden im flüssigen Stickstoff in der Medienküche aufbewahrt. Die Zellsammlung wird von Susanne verwaltet.

Virensammlung: umfasst alle lentiviralen, adenoviralen, AAV-Partikel und Bakteriophagen. Werden bei -70°C aufbewahrt. Access-Datei!

Oligosammlung: umfasst alle Oligos, die für Klonierungen, PCRs, etc. bestellt wurden. Die Oligos werden als 10 mM Stammlösung in TE-Puffer bei -20°C in der allgemeinen Sammlung, nicht in privaten Gefrierschränken, aufbewahrt. Bestellung neuer Oligos und Ablage der Informationen erfolgt über Jan Kuiper.

Antikörper-Sammlung: Sammlung aller Primärantikörper (kommerziell oder selbst hergestellt) und Sekundärantikörper (Enzym- oder Fluoreszenz-konjugiert). Datenblätter werden im Ordner „Antikörper“ gesammelt. Claudia Hentschel organisiert die Antikörper-Sammlung.

Protein-Sammlung: Hier werden alle rekombinanten, gereinigten Proteine erfasst.

Inhibitor-Sammlung: Hier werden Informationen zu allen pharmakologischen Inhibitoren und die Konzentration, das Lösungsmittel und der Aufbewahrungsort von Stammlösungen abgelegt. _______________ organisiert die Inhibitor-Sammlung.

Enzym-Sammlung: umfasst alle kommerziellen und selbst hergestellten (DpnI) Restriktionsenzyme, die bei -20°C gelagert werden. Entnahme von Enzymen aus den Vorratsgefäßen nur mit FILTERSPITZEN!

Die Enzyme dürfen nur für eine möglichst kurze Zeitspanne (1-2 Minuten) aus dem Gefrierschrank entnommen werden (d.h. direkt vor Gebrauch) und werden während dieser kurzen Zeit in einem auf -20°C-gekühlten Paraffinblock gelagert.

''Ordner mit Dokumentation zu allen Sammlungen finden sich in den Regalen am allgemeinen Arbeitsplatz im Bürobereich.''

== '''Kompetente Zellen''' ==
Aliquots kompetenter Bakterien werden bei -80°C gelagert. Je Aliquot sind 200 µl weggefroren, ausreichend für 2 Transformationen. Verschiedene Stämme stehen für unterschiedliche Zwecke zur Verfügung. Der Standardklonierungsstamm ist NovaBlue. Bei der Entnahme möglichst zügig vorgehen, Erwärmung der Aliquots führt zu verringerter Kompetenz, nicht mehr Aliquots entnehmen als tatsächlich gebraucht werden.

== '''Enzyme''' ==
Polymerasen, Restriktionsenzyme, etc. werden bei -20°C in Boxen bzw. auf Paraffinblöcken gemeinsam mit den entsprechenden Puffern aufbewahrt. Diese Enzyme werden von Allen gemeinsam benutzt. Deshalb besonders reinlich mit den Enyzm-Stammlösungen umgehen und Verunreinigungen (Filterspitzen !!!) sowie Erwärmung vermeiden.

Bei der Entnahme von Enzymen, die Eppis mit der Stammlösung ausschließlich in einem auf -20°C-gekühlten Paraffinblöckchen zum Arbeitsplatz transportieren und darin zum Pipettieren belassen. Auch mit Paraffinblock möglichst das Enzym nur kurzzeitig aus dem -20°C Gefrierschrank entnehmen (< 2 Minuten) und sofort nach dem Pipettieren wieder zurückbringen. Beim Öffnen der Vorratsgefäße auf jeden Fall Kontakt der Deckelinnenseite mit der Haut vermeiden!!

== '''Bestellungen''' ==
Wenn allgemeine Chemikalien oder Einwegartikel zur Neige gehen, RECHTZEITIG (d.h. vor dem Aufbrauchen der letzten Packung oder dem Abpipettieren des letzten Mikroliters) Bescheid geben: entweder Anschreiben an die Tafel im Labor oder den Bestellern sagen. Projektspezifische Chemikalien/Antikörper etc. oder Laborutensilien/-geräte vor Bestellung mit Christof absprechen.

Für Oligobestellungen über Jan die Oligo-Informationen in die Bestell-Liste auf dem Fileserver eintragen: Fileserver_LSH:\Stammsammlung\Primer\01_PrimerOrdering_Primerbestellung

Andere on-line Bestellungen mit Christof absprechen.

Bestellung von Sequenzierungen über pre-paid labels von LGC oder GeneWiz. Bitte nicht mehr als 2 Sequenzierung pro neuem Konstrukt. Größeren Sequenzierbedarf mit Christof absprechen. Lieferungen von Reagenzien sollen dem Besteller übergeben werden. Besteller prüft auf Vollständigkeit (unter Nutzen des Lieferscheins) und bestätigt mit Datum und Unterschrift. Der Lieferschein und eventuell beiliegende Rechnungen werden im Sekretariat an Petra Schnurr weitergegeben. Bei Lieferung von Chemikalien, Antikörpern, Enzymen und anderen Reagenzien werden die Datenblätter mit einem Datum versehen und in den entsprechenden Ordnern abgelegt (s. „Allgemeine Sammlungen“). Auf den Datenblättern wird eventuell das Lösungsmittel, die Konzentration und eventuell abgefüllte Aliquots sowie der Lagerort vermerkt.

ALLE Chemikalien, Reagenzien, Enzyme, etc. werden an zugänglichen und bekannten Orten gelagert.

== '''Laborsicherheit''' ==
Siehe dazu [[All About Safety|Safety Guidelines]]

Lab Etiquette

2025-06-05T12:14:00Z

ESO wikiadmin: /* Laborbuch */

'''Diese Seite ist auch auf [[Lab Etiquette (ENG)|Englisch 🇬🇧]] verfügbar.'''

== '''Laborzugang''' ==
Das Laborgebäude ML ist zwischen 6:30 und 20:30 Uhr an Werktagen geöffnet. Zugang zu den Laborräumen erhält man mittels RFID-Transponder, der es auch erlaubt, das Laborgebäude außerhalb der oben stehenden Öffnungszeiten zu betreten. Masterstudenten und Doktoranden erhalten einen persönlichen Transponder über die Schlüsselausgabe (Formular bei Petra Schnurr) und müssen den Transponder nach Beendigung ihrer Zeit als Mitarbeiter wieder abgeben. Für Bachlorstudenten und Hiwis gibt es nach Einarbeitung in Ausnahmefällen einen Leihtransponder für notwendige Arbeiten außerhalb der Öffnungszeiten. Der Leihtransponder befindet sich im Schlüsselkasten am allg. Computer-Arbeitsplatz. Die Entnahme des Leihtransponders ist in der Liste mit Datum und Kürzel zu vermerken.

== '''Arbeitszeiten''' ==
Nicht-wissenschaftliche Mitarbeiter und Hiwis sind verpflichtet, die Arbeitszeit zu erfassen. Hierzu steht den Mitarbeitern die Online-Webseite der Personalverwaltung zur Verfügung:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

Hiwis nutzen zur Zeiterfassung den Stundenzettel (erhältlich bei Petra Schnurr)

Wissenschaftliche Mitarbeiter sollen zu den Kernarbeitszeiten 9.00 – 12.00 und 14.00 – 16.00 anwesend sein. Eine Abwesenheit ist mit Christof abzusprechen und im Wandkalender im Eingangsbereich der Bürozeile zu vermerken.

== '''Krankheit''' ==
Im Krankheitsfall soll so bald wie möglich Christof und das Sekretariat per Telefon und/oder E-Mail benachrichtigt werden, damit Vertretung, etc. organisiert werden kann. Bei längerer Krankheit muss am dritten Tag der Abwesenheit eine ärztliche Krankmeldung/ein ärztliches Attest an petra.schnurr@uni-konstanz.de geschickt werden.

== '''Urlaub''' ==
Mitarbeiter beantragen möglichst frühzeitig (mindestens 1 Woche vorher) ihren Urlaub über das online Zeiterfassungssystem:

''<nowiki>https://personal.uni-konstanz.de/qisserver/rds?state=user&type=0&topitem=</nowiki>''

Die Möglichkeit für längerfristigen Urlaub (> 2 Wochen) ist projektabhängig und soll mindestens 2 Monate vorher mit Christof abgesprochen werden. In der 2.Hälfte des Wintersemesters (Anfang Januar – Ende Februar) ist während des VTK Urlaub nur in Ausnahmefällen möglich.

Vor Urlaubsantritt muss immer die Kultur/Lagerung von wichtigen Klonen/Zellinien/Proben sichergestellt und eine Vertretung für den Labjob organisiert sein. Bachelor-/Masterstudenten sprechen eine mehrtägige Abwesenheit mit Ihrem/Ihrer BetreuerIn und Christof ab.

== '''Seminare/Projektbesprechungen''' ==
Montags vormittags (8.45 Uhr) ist eine allg. Laborbesprechung. Im Anschluß daran eine Porjektvorstellung einzelner Mitarbeiter, die am Notice-Board im Eingangsbereich angekündigt wird.

Mittwochs am Nachmittag von 15.15 Uhr – ca. 17.00 Uhr findet unser Journal-Club statt. Die jeweils behandelte Publikation wird in der Woche zuvor auf den Fileserver geladen bzw. als pdf-file an alle verschickt. Im 14-tägigen Turnus besprechen wir in diesem Seminar aktuelle neue Daten aller Mitarbeiter.

Die Teilnahme an diesen beiden Seminaren ist obligatorisch, der Seminarplan wird in der Regel auf ein viertel- bis ein halbes Jahr im voraus festgelegt.

Dienstags nachmittags und donnerstags zur Mittagszeit finden während der Vorlesungszeit die Seminare der Graduiertenschule KoRS-CB sowie das Fachbereichsseminar des FB Biologie statt. Die Vortragenden/Themen werden durch Aushang bekannt gegeben. Die Teilnahme ist fakultativ, aber wenn möglich sollte man sich die interessanten Vorträge nicht entgehen lassen.

Spezifische Projektbesprechungen finden nach Absprache mit Christof statt. Zu den Projektbesprechungen ist das Laborbuch als Gesprächsgrundlage mitzubringen.

== '''Namenskürzel/Beschriftungen''' ==
Jeder Mitarbeiter benutzt ein unverwechselbares Namenskürzel, mit dem alle Proben, Lösungen, etc. beschriftet werden müssen. Die Namenskürzel werden im Sekretariat vergeben und sind in einer Liste auf dem Fileserver abgelegt:

Fileserver_LSH/Vorlagen/Labor-Verzeichnisse/Namenskuerzel_AG Hauck_2025.doc

== '''Laborbuch''' ==
Jeder Mitarbeiter führt ein Laborbuch, das im Sekretariat ausgegeben wird und in dem alle Arbeiten in chronologischer Reihenfolge dokumentiert werden. Muster für die Dokumentation von verschiedenen Experimenten sind als pdf-file auf dem Fileserver abgelegt:

Fileserver_LSH/Vorlagen/Labor-Protokollierung/

* Gentamicin-Assay: „Steffi“
* PCR: „''PCR-based-LIC-cloning-protocol-CRH.pdf''“
* Zelltransfektion: „''HEKcell-transfection-Protocol-JWK.pdf''“
* In vitro Kinase Assay: “Erik”
* [[Immunoprecipitation|Immunopräzipitation]] (MHE)
* Protein expression and purification: “Sarah”
* Lentivirus production: “Marlene”
* Bacterial growth curve: “Ann-Kathrin”

Die Seiten des Laborbuchs werden durchnumeriert und auf den ersten beiden Seiten wird ein Inhaltsverzeichnis angelegt.

Das Laborbuch gehört dem Lehrstuhl Zellbiologie und verbleibt nach dem Ausscheiden im Besitz des Lehrstuhls. Aus dem Laborbuch dürfen keine Seiten entfernt werden. Originalausdrucke, Kopien von Gelen, Röntgenfilme, etc. werden beschriftet und dem Laborbuch beigelegt, indem sie zu den entsprechenden Experimenten geheftet oder geklebt werden. Die elektronischen Originaldaten werden auf dem Fileserver in einem eigenen User-Verzeichnis gespeichert (s. Kapitel Daten Archivierung).

Für die meisten Experimente gibt es bereits Labor-Protokolle, die in unserem VTK-Skript ausführlich beschrieben sind und auf die im Laborbuch verwiesen werden kann. Das VTK-Skript findet sich auf dem Fileserver:

''Fileserver_LSH/Vorlagen/Labor-Protokollierung/Skript-Vertiefungskurs-2025.doc''

Die verwendeten Reagenzien und Lösungen sind ebenfalls entweder in der Rezepte-Datei beschrieben: ''Fileserver_LSH/Rezepte/Rezepte-2025.doc'' oder sind in der Chemikalienliste zu finden:

''Fileserver_LSH/Chemikalienliste/Chemikalienliste 2025 Gruppe Hauck.xls''

Nur bei Abweichen vom existierenden Protokoll sind besondere Vermerke im Laborbuch notwendig.

Bei mikroskopischen Arbeiten, durchflußzytometrischen Analysen oder ähnlichen Arbeiten mit Ergebnissen in elektronischer Form wird ein Probenprotokoll angefertigt (s. Fileserver_LSH/Vorlagen/Labor-Protokollierung/Protokoll-Mikroskop-FACS.doc“) und dem Experiment beigeheftet.

Bei Projektbesprechungen dient das Laborbuch als Grundlage für die Diskussion der Ergebnisse und ist deshalb unbedingt mitzubringen.

== '''Daten-Archivierung''' ==
Standardexperimente sollen anhand der oben angeführten Laborprotokolle durchgeführt werden, Abweichungen sind im Laborbuch festzuhalten. Die im Rahmen der Experimente erhaltenen Rohdaten (z.B. Mikroskopiebilder, Gelbilder, FACS-Daten, VarioScan-Daten) sowie die daraus hergestellten abgeleiteten Abbildungen (Graphiken, berbeiteten Mikroskopiebilder, etc.) sind auf dem Daten-Fileserver „Fileserver_LSH_Data/00_User/„ in einem separaten Nutzerfolder zu speichern. Nur dort erfolgt eine automatische (tägliche) Sicherung, so dass keine Daten verloren gehen können.

Jede elektronische Datendatei trägt im Namen das Datum, das Namenskürzel und das Experiment (The raw data files are named according to the following scheme):

'''Jahr-Monat-Tag_Namenskürzel_Experiment / year-month-day_initials_experiment'''

z.B. /e.g.: 2025-01-01_CRH_Adhesion assay

- the digital version/raw files should be on the file server with the date that can also be found in the lab book

- a printed version of the evaluated data should be attached in the lab book

For later use in publications it is important that we all stick to the same folder structure and naming of files. An example of such a user folder is given on the Fileserver and depicted on the next page:

Fileserver_LSH_Data/00_User/xUE_Universal_Example/

'''Data'''

'' Constructs''

SuperSH2 agarose gels, sequencing results, plasmid map, etc.

'' Western Blots''

'' Immunofluorescence staining''

'' Flow Cytometry''

'' ELISA/SEAP-Assays''

''' .'''

''' .'''

'''Literature'''

'''Presentations'''

'''Thesis:''' for your thesis: for '''every''' figure in your thesis create a subfolder named with the Figure-Number and include in each subfolder both: the final figure as pdf- and illustrator-file and the '''used raw data''' (subdivided in panels A, B, etc.). The name of the raw data file should remain the same as it is found in your DATA-folder and lab book)
[[File:Labetiquette fileserver.jpg|center|thumb|700x700px|'''Fig. 1:''' Example of the user folder structure on the Fileserver_LSH_Data of user UE_Universal_Example.]]

== '''Laborarbeitsplatz''' ==
In den Laboren gibt es allgemeine Laborarbeitsplätze (z.B. in den beiden Zellkulturlaboren, im Infektionsraum oder der Bereich mit Proteingel-Elektrophorese) sowie private Arbeitsplätze, die zugewiesen sind. Arbeiten auf den privaten Arbeitsplätzen anderer soll nur nach Rücksprache erfolgen. Jeder ist für die Sauberhaltung seines Arbeitsplatzes, der dazugehörigen Regale und Schubladen selbst verantwortlich. Die Arbeitsfläche wird mindestens einmal pro Woche abgewischt und desinfiziert, die Schubladen/Regale sind regelmäßig abzustauben und ordentlich zu halten.

Zusätzlich hat jeder einen Lagerplatz in einem Kühlschrank bei 4°C, in einem Gefrierschrank/-fach bei -20°C und kann Proben in beschrifteten Boxen im -80°C-Schrank in Raum ML lagern. Für die Lagerung stehen Kunststoffboxen mit Deckel zur Verfügung, offenen Ständer (speziell für Eppendorfgefäße) sollen nur für Kurzzeit-Aufbewahrung (1 Tag) verwendet werden. Alle Proben (Eppis, Agarplatten, etc.) müssen mit Kürzel, Inhaltsbezeichnung und Datum beschriftet sein. Alle Lagerplätze sollen regelmäßig (mindestens 1x je Monat) aufgeräumt/aktualisiert werden. Bei Lagerung im -80°C Schrank ist für jede Box ein Datenblatt in den -80°C-Ordner abzulegen. Eine Vorlage für das Datenblatt findet sich auf dem Fileserver:

''Fileserver_LSH/Vorlagen/Labor-Protokollierung/'' ''-80C Box-Vorlage.doc''

Das Öffnen von Tiefgefrierschränken bitte auf ein Minimum reduzieren (da sonst Vereisung und Fehlfunktion). Deshalb bereits VOR dem Öffnen überlegen, was entnommen werden soll und wo es zu finden ist (s. Datenblatt!!!). Allgemeine Laborarbeitsplätze sind nach jeder Benutzung zu säubern (z.B. sterile Werkbank, Proteingel-Elektrophorese, Abzug, Waagen, Geldoku) und alle verwendeten Lösungen, Apparaturen, etc. müssen wieder aufgeräumt werden.

== '''Lab-Jobs''' ==
Jeder Mitarbeiter erhält einen Labjob, den er für die Allgemeinheit wahrnimmt. Der Lab-Job kann die Pflege einer allgemeinen Sammlung, die Verantwortung für ein Gerät oder die Aufsicht und Organisation eines Labors beinhalten. Darüber hinaus wird die Herstellung von Lösungen/Puffern etc. für die Allgemeinheit über Lab-Jobs. Die jeweils Verantwortlichen sind auf einer Liste am am Noticeboard am Eingang aufgeführt. Die Raum-Verantwortlichen organisieren auch die regelmäßige Pflege/Putzpläne für allgemeine Geräte wie sterile Werkbänke, Inkubatoren, etc.

== '''Schließdienst''' ==
Der abendliche Schließdienst, der dafür sorgt, dass Geräte wie sterile Werkbänke, Vakkumpumpen, Geldoku oder die Propangasleitung ausgeschaltet sind, wechselt im wöchentlichen Turnus unter den wissenschaftlichen Mitarbeitern und Masterstudenten. Eine Liste für den abendlichen Kontrollgang des Schließdienstes hängt am Noticeboard am Eingang.

== '''Computernutzung''' ==
Im Seminarraum befindet sich ein Computer zur allgemeinen Nutzung. Bitte jederzeit die Nutzung für dienstliche Zwecke ermöglichen. Private PCs/Notebooks können an das Netzwerk angeschlossen werden. Voraussetzung ist ein aktueller Virenscanner und die Aktivierung einer Firewall. Den Zugang zu den Netzwerk-Speichern des Lehrstuhls im Rechenzentrum (Fileserver_LSH und Fileserver_LSH_Data) richtet Christof ein. Auf dem Fileserver_LSH_Data/02_SOFTWARE stehen verschiedene Software-Programme zur Verfügung, darunter Adobe Photoshop CS4, Adobe Acrobat Pro9, Adobe Illustrator CS4, MS Office 2016, Clone Manager 9, Endnote X9, SigmaStat 4.0, Leica LAS, Biorad QuantityOne, VarioScan SkanIt. Um den problemlosen Austausch der Daten innerhalb des Labors zu gewährleisten, müssen die oben angegebenen Versionen dieser Programme für alle dienstlichen Zwecke verwendet werden bzw. die Daten in einer mit diesen Programmversionen kompatiblen Form gespeichert werden.

== '''Die Mitarbeit jedes Einzelnen ist gefragt''' ==
Jeder Mitarbeiter kann durch wenig Aufwand dazu beitragen, dass sich die Zusatzarbeiten im Labor für Alle minimieren. So soll jeder durch vorausschauende Planung den anfallenden Verbrauch/Abfall/Aufräumaufwand möglichst gering halten und auch bei der Entsorgung von Abfällen mithelfen. Bitte Autoklaviersäcke nicht bis zum „Überquellen“ füllen, sondern rechtzeitig verschließen, in der Wanne deponieren und neuen Autoklaviersack in den entsprechenden Müllbehälter geben.

Jeder ist auch selbst für das Befüllen der Boxen mit Pipettenspitzen und das Auffüllen von Eppis verantwortlich. Autoklaviergut wird in der Plastikkiste gesammelt und von Claudia/Susanne für alle autoklaviert.

Zum verträglichen Umgang gehört auch das Verhalten und die sauberkeit in der Teeküche und im Sozialraum. Wenn ich im Verlaufe von 1 Monat nie die Spülmaschine ein- oder ausgeräumt oder die Kaffeemaschine aufgefüllt habe, dann habe ich mich sehr wahrscheinlich auf Kosten von Anderen am sauberen Geschirr bedient.

== '''Chemikalien/Lösungen''' ==
Viele Lösungen werden als Lab-Job angesetzt und entweder als Stocklösung an festgelegten Plätzen für Alle zugänglich aufbewahrt oder als aliquotierte Lösung bereitgestellt, wobei man sich bei Bedarf ein Aliquot an den eigenen Arbeitsplatz mitnimmt und dort für die weitere Verwendung aufbewahrt. Weitere Lösungen für den eigenen Bedarf sind selbst anzusetzen (bitte die benötigten Mengen im Voraus abschätzen und nicht Liter-weise Lösungen ansetzen, wenn man für zwei Experimente insgesamt nur 10 ml braucht) und korrekt beschriftet (Kürzel, Datum, Inhalt: Bezeichnung und Konzentration; Gefahrensymbol) am eigenen Arbeitsplatz aufzubewahren. Lösungen und Aliquots am Arbeitsplatz der Kollegen NUR nach Rücksprache benutzen. Stocklösungen, aliquotierte Lösungen und die jeweiligen Verantwortlichen sind auf der Liste am Schwarzen Brett „Lab-Jobs“ aufgeführt. Für das Ansetzen von Lösungen gibt es ein Rezeptbuch, das als Datei auf dem Fileserver bereit liegt:

Fileserver_LSH//Rezepte/ Rezepte-2025.doc

== '''Allgemeine Sammlungen''' ==
Der Lehrstuhl unterhält verschiedene Sammlungen, die eine wichtige Ressource für Alle darstellen und mit denen deshalb besonders umsichtig und verantwortlich umgegangen werden muss.

Stammsammlung: umfasst alle Bakterienstämme, die nach Nummern abgelegt sind. Lagerung bei -70°C in Truhe auf Ebene 5 sowie in Raum ML6 :

– nichtpathogene Bakterien (=gleichzeitig Repositorium aller hergestellten Plasmide)

– pathogene Bakterien und Mutanten (Schlüssel dafür bei Christof)

Die Stammsammlung ist auch als MS Access-Datei vorhanden. Jeder neu hergestellte oder von extern erhaltene Bakterienstamm erhält eine neue Stammnummer und wird in der Stammsammlung eingefroren sowie elektronisch erfasst. Die Verteilung der Nummern erfolgt über Christof. Zugang zur Stammsammlung haben nur Susanne und Christof.

Plasmidsammlung: umfasst alle isolierten Plasmide aus den nichtpathogenen Bakterienstämmen der Stammsammlung. Die Numerierung ist identisch mit der Stammsammlung (Lagerung im allg. Kühlschrank bei 4°C)

Zellsammlung: umfasst Stocks aller Zellinien und primärer Zellen. Werden im flüssigen Stickstoff in der Medienküche aufbewahrt. Die Zellsammlung wird von Susanne verwaltet.

Virensammlung: umfasst alle lentiviralen, adenoviralen, AAV-Partikel und Bakteriophagen. Werden bei -70°C aufbewahrt. Access-Datei!

Oligosammlung: umfasst alle Oligos, die für Klonierungen, PCRs, etc. bestellt wurden. Die Oligos werden als 10 mM Stammlösung in TE-Puffer bei -20°C in der allgemeinen Sammlung, nicht in privaten Gefrierschränken, aufbewahrt. Bestellung neuer Oligos und Ablage der Informationen erfolgt über Jan Kuiper.

Antikörper-Sammlung: Sammlung aller Primärantikörper (kommerziell oder selbst hergestellt) und Sekundärantikörper (Enzym- oder Fluoreszenz-konjugiert). Datenblätter werden im Ordner „Antikörper“ gesammelt. Claudia Hentschel organisiert die Antikörper-Sammlung.

Protein-Sammlung: Hier werden alle rekombinanten, gereinigten Proteine erfasst.

Inhibitor-Sammlung: Hier werden Informationen zu allen pharmakologischen Inhibitoren und die Konzentration, das Lösungsmittel und der Aufbewahrungsort von Stammlösungen abgelegt. _______________ organisiert die Inhibitor-Sammlung.

Enzym-Sammlung: umfasst alle kommerziellen und selbst hergestellten (DpnI) Restriktionsenzyme, die bei -20°C gelagert werden. Entnahme von Enzymen aus den Vorratsgefäßen nur mit FILTERSPITZEN!

Die Enzyme dürfen nur für eine möglichst kurze Zeitspanne (1-2 Minuten) aus dem Gefrierschrank entnommen werden (d.h. direkt vor Gebrauch) und werden während dieser kurzen Zeit in einem auf -20°C-gekühlten Paraffinblock gelagert.

''Ordner mit Dokumentation zu allen Sammlungen finden sich in den Regalen am allgemeinen Arbeitsplatz im Bürobereich.''

== '''Kompetente Zellen''' ==
Aliquots kompetenter Bakterien werden bei -80°C gelagert. Je Aliquot sind 200 µl weggefroren, ausreichend für 2 Transformationen. Verschiedene Stämme stehen für unterschiedliche Zwecke zur Verfügung. Der Standardklonierungsstamm ist NovaBlue. Bei der Entnahme möglichst zügig vorgehen, Erwärmung der Aliquots führt zu verringerter Kompetenz, nicht mehr Aliquots entnehmen als tatsächlich gebraucht werden.

== '''Enzyme''' ==
Polymerasen, Restriktionsenzyme, etc. werden bei -20°C in Boxen bzw. auf Paraffinblöcken gemeinsam mit den entsprechenden Puffern aufbewahrt. Diese Enzyme werden von Allen gemeinsam benutzt. Deshalb besonders reinlich mit den Enyzm-Stammlösungen umgehen und Verunreinigungen (Filterspitzen !!!) sowie Erwärmung vermeiden.

Bei der Entnahme von Enzymen, die Eppis mit der Stammlösung ausschließlich in einem auf -20°C-gekühlten Paraffinblöckchen zum Arbeitsplatz transportieren und darin zum Pipettieren belassen. Auch mit Paraffinblock möglichst das Enzym nur kurzzeitig aus dem -20°C Gefrierschrank entnehmen (< 2 Minuten) und sofort nach dem Pipettieren wieder zurückbringen. Beim Öffnen der Vorratsgefäße auf jeden Fall Kontakt der Deckelinnenseite mit der Haut vermeiden!!

== '''Bestellungen''' ==
Wenn allgemeine Chemikalien oder Einwegartikel zur Neige gehen, RECHTZEITIG (d.h. vor dem Aufbrauchen der letzten Packung oder dem Abpipettieren des letzten Mikroliters) Bescheid geben: entweder Anschreiben an die Tafel im Labor oder den Bestellern sagen. Projektspezifische Chemikalien/Antikörper etc. oder Laborutensilien/-geräte vor Bestellung mit Christof absprechen.

Für Oligobestellungen über Jan die Oligo-Informationen in die Bestell-Liste auf dem Fileserver eintragen: Fileserver_LSH:\Stammsammlung\Primer\01_PrimerOrdering_Primerbestellung

Andere on-line Bestellungen mit Christof absprechen.

Bestellung von Sequenzierungen über pre-paid labels von LGC oder GeneWiz. Bitte nicht mehr als 2 Sequenzierung pro neuem Konstrukt. Größeren Sequenzierbedarf mit Christof absprechen. Lieferungen von Reagenzien sollen dem Besteller übergeben werden. Besteller prüft auf Vollständigkeit (unter Nutzen des Lieferscheins) und bestätigt mit Datum und Unterschrift. Der Lieferschein und eventuell beiliegende Rechnungen werden im Sekretariat an Petra Schnurr weitergegeben. Bei Lieferung von Chemikalien, Antikörpern, Enzymen und anderen Reagenzien werden die Datenblätter mit einem Datum versehen und in den entsprechenden Ordnern abgelegt (s. „Allgemeine Sammlungen“). Auf den Datenblättern wird eventuell das Lösungsmittel, die Konzentration und eventuell abgefüllte Aliquots sowie der Lagerort vermerkt.

ALLE Chemikalien, Reagenzien, Enzyme, etc. werden an zugänglichen und bekannten Orten gelagert.

== '''Laborsicherheit''' ==
Siehe dazu [[All About Safety|Safety Guidelines]]

'''Schutzkleidung'''

'''In den Laboren ist bei allen Arbeiten IMMER ein geschlossener Labormantel und eine Schutzbrille zu tragen. Besondere Vorsicht ist bei Arbeiten mit flüssigem Polyacrylamid (Gießen von SDS-PAGE-Gelen) und bei Arbeiten mit Säuren/Laugen walten zu lassen. Zusätzlich zu Labormantel und Schutzbrille sind hierbei Handschuhe zu tragen.'''

'''Gas'''

'''Laborgas ist zentral in Raum ML in einem Sicherheitsschrank untergebracht. Die Gasversorgung soll abends durch Drehen des Schlüssels abgestellt werden. An den Arbeitsplätzen ist nach Beendigung der Arbeiten mit Gas der Absperrhahn zu schließen sind.'''

'''Scharfe Gegenstände:'''

'''- Kanülen/Rasierklingen/Skalpelle'''

'''Vorsicht bei der Arbeit mit diesen Utensilien: Verletzungsgefahr! Kanülen nach Gebrauch nicht mehr in die Plastikhülle zurückstecken, sondern entsorgen.'''

'''Diese Materialien werden nach Gebrauch in entsprechend beschrifteten, gelben Kunststoff-Behältern (Einweg-Behälter) gesammelt. Einer dieser Behälter steht im Kühlraum, ein zweiter solcher Behälter steht unter Abzug II.'''

'''- Pasteurpipetten'''

'''Pasteurpipetten werden in den großen, gelben Kunststoff-Behältern (Mehrweg) gesammelt, die mit „Nur Pasteurpipetten/''Pasteur-Pipetts ONLY''“ beschriftet sind. Je ein Behälter dieser Art steht in den Zellkulturlabors und im Kühlraum. Bitte KEINE anderen scharfen Gegenstände (Skalpelle, Rasierklingen, Kanülen, etc.) in diese Behälter werfen, da der Inhalt nach Autoklavieren in den Glasabfall wandert.'''

'''- Glasflaschen/Glasbruch'''

'''Glasbruch und leere Glasflaschen mit Laborglas werden in den gekennzeichneten Mülleimern gesammelt.'''

'''Einweisung dueh Raumverantwortliche'''

'''Arbeiten im Zelllabor'''

'''Fluoreszenzmikroskopxxx'''

'''Arbeiten im S2-Labor (Infektionslabor)'''

'''xxx'''

'''Arbeiten am RNA-Arbeitsplatz'''

'''xxx'''

'''Mikroskopie - Marleen'''

'''Varioscan – Erik'''

'''Casy-Zellzähler'''

'''TFA'''

'''Unfälle'''

'''Beim ersten Aufenthalt im Labor mit den Schutzeinrichtungen (Notfallduschen, Augenduschen, Feuerlöscher, Sicherheitsschalter), Notfallnummern (Am Telefon neben dem Aufzug), Erste Hilfe Kasten (ebenfalls am Telefon neben dem Aufzug) und Fluchtwegen vertraut machen.'''

'''Bei Kontakt der Augen mit Chemikalien/Krankheitserregern:'''

'''Sofort Ausspülen der Augen mit Hilfe der Augenduschen, dann die Ersthelfer (Tel.-Nr. 2222) benachrichtigen lassen.'''

'''Bei Kontakt der Haut/Laborkleidung mit Chemikalien, bei Brand der Laborkleidung:'''

'''Notfallduschen im Gang oder in Labor nutzen'''

'''Verletzungen/Schnitte:'''

'''Ersthelfer kontaktieren'''

'''Bei Ausschütten von Lösungen/Krankheitserregern: sofort Unfallstelle kennzeichnen und absichern, dann erst mit Inaktivierung/Dekontamination beginnen'''

'''Feuer'''

'''Fluchtwege und Sammelpunkt einprägen'''

'''Zentrifugen'''

'''Die Bedienung der Zentrifugen darf erst nach Einweisung durch die für Zentrifugen verantwortliche Person erfolgen (Susanne Feindler-Boeckh). Immer Zentrifugengefäße austarieren, bei vielen Zentrifugen stehen entsprechend befüllte Tara-Röhrchen bereits bereit. Nicht alle Zentrifugengefäße passen in jeden Rotor. Bitte auf richtige Passung achten und nie Gewalt anwenden, um Zentrifugenbecher in den Rotor zu laden.'''

'''Ultrazentrifugenrotoren immer mit ALLEN Gehängen in der vorgesehenen Position betreiben.'''

'''Bei der Zentrifugation von Suspensionen humanpathogener Bakterien sind die besonderen Betriebsanweisungen zu beachten.'''

'''Ethidiumbromid'''

'''DNA-Färbung von Agarosegelen ausschließlich in der Dunkelkammer in dem dafür vorgesehen Färbe/Entfärbebad durchführen. Dort stehen auch Nitrilhandschuhe bereit, die nur für diesen Zweck und NICHT außerhalb der Dunkelkammer verwendet werden. EINE behandschuhte Hand reicht aus, so dass Ethidiumbromid-freie Gegenstände (z.B. Hauptschalter der Steckdose) mit der Handschuh-freien Hand bedient werden können.'''

'''Wasser für das Entfärbebad ist regelmäßig zu erneuern (mindestens täglich). Die Entfärbelösung kann ins Waschbecken entleert werden und durch normales VE-Wasser aufgefüllt werden.'''

'''Gefahrstoffe'''

'''Bei der Arbeit mit Gefahrstoffen sind die gültigen Sicherheitsbestimmungen einzuhalten. Insbesondere gilt bei der Arbeit mit:'''

'''''Paraformaldehyd'': ist im ungelösten Zustand unter dem Abzug abzuwiegen.'''

'''''Mercaptoethanol-Lösungen'': sind nur unter dem Abzug zu handhaben.'''

'''''Brennbare und leicht flüchtige Substanzen (Methanol, Ethanol)'': Die Vorräte sind im Sicherheitsschrank aufzubewahren. Geringe Mengen (Tagesbedarf) können am Arbeitsplatz in gekennzeichneten Gefäßen aufbewahrt werden.'''

'''''Phenol und Phenolhaltige-Lösungen'': dürfen nur unter Abzug II gehandhabt werden. Dort sind auch Abfallbehälter für die Entsorgung von flüssigen bzw. festen/brennbaren Phenolhaltigen Abfällen.'''

'''Acrylamid'''

'''Abfälle'''

'''Abfälle von giftigen Chemikalien, Farbstoffen, Fixantien, werden separat in beschrifteten Abfallkanistern und –flaschen gesammelt, bevor sie sachgemäß über das Chemiklaien-Abfalllager entsorgt werden.'''

'''Betriebsärztliche Untersuchung'''

'''Jeder Mitarbeiter muss 1x/Jahr vom Betriebsarzt untersucht werden. Dazu wird bei Einstellung/Arbeitsbeginn ein Termin über Petra Schnurr organisiert. Die weiteren jährlichen Untersuchungen sind selbständig zu organisieren und eine Kopie der Unbedenklichkeitserklärung des Betriebsarztes ist nach erfolgter Untersuchung im Sekretariat bei Petra Schnurr abzugeben.'''

'''Sicherheitsbelehrung'''

'''Eine allg. Sicherheitsbelehrung zur biologischen Sicherheit führt Stefan Kreft, Beauftragter für Biol. Sicherheit der Universität, 1-mal jährlich durch. Im Zusammenhang mit dem VTK gibt es zusätzlich 1-mal jährlich eine arbeitsplatzspezifische Sicherheitsbelehrung. Beide Veranstaltungen sind obligatorisch.'''

'''Bei Arbeitsbeginn ist ebenfalls eine Sicherheitsbelehrung und Laboreinweisung zu absolvieren.'''

Laborsicherheit

2025-06-05T11:59:57Z

ESO wikiadmin:

Diese Seite ist auch in [[All About Safety|Englisch 🇬🇧]] verfügbar.

== '''Schutzkleidung''' ==
In den Laboren ist bei allen Arbeiten IMMER ein geschlossener Labormantel und eine Schutzbrille zu tragen. Besondere Vorsicht ist bei Arbeiten mit flüssigem Polyacrylamid (Gießen von SDS-PAGE-Gelen) und bei Arbeiten mit Säuren/Laugen walten zu lassen. Zusätzlich zu Labormantel und Schutzbrille sind hierbei Handschuhe zu tragen.

== '''Gas''' ==
Laborgas ist zentral in Raum ML in einem Sicherheitsschrank untergebracht. Die Gasversorgung soll abends durch Drehen des Schlüssels abgestellt werden. An den Arbeitsplätzen ist nach Beendigung der Arbeiten mit Gas der Absperrhahn zu schließen sind.

== '''Scharfe Gegenstände:''' ==

* '''Kanülen/Rasierklingen/Skalpelle''' Vorsicht bei der Arbeit mit diesen Utensilien: Verletzungsgefahr! Kanülen nach Gebrauch nicht mehr in die Plastikhülle zurückstecken, sondern entsorgen. Diese Materialien werden nach Gebrauch in entsprechend beschrifteten, gelben Kunststoff-Behältern (Einweg-Behälter) gesammelt. Einer dieser Behälter steht im Kühlraum, ein zweiter solcher Behälter steht unter Abzug II.

* '''Pasteurpipetten''' Pasteurpipetten werden in den großen, gelben Kunststoff-Behältern (Mehrweg) gesammelt, die mit „Nur Pasteurpipetten/''Pasteur-Pipetts ONLY''“ beschriftet sind. Je ein Behälter dieser Art steht in den Zellkulturlabors und im Kühlraum. Bitte KEINE anderen scharfen Gegenstände (Skalpelle, Rasierklingen, Kanülen, etc.) in diese Behälter werfen, da der Inhalt nach Autoklavieren in den Glasabfall wandert.

* '''Glasflaschen/Glasbruch''' Glasbruch und leere Glasflaschen mit Laborglas werden in den gekennzeichneten Mülleimern gesammelt.

== '''Einweisung dueh Raumverantwortliche''' ==
'''Arbeiten im Zellkulturlabor – Jedes neue Mitglied muss die Einführung in das Zellkulturlabor durch SSJ bestehen, bevor es dort arbeiten kann. Weitere Informationen hier: [[S1 - Cell culture guidelines]]'''

Fluoreszenzmikroskopxxx

'''Arbeiten im S2-Labor (Infektionslabor)'''

xxx

'''Arbeiten am RNA-Arbeitsplatz'''

xxx

'''Mikroskopie - Marleen'''

'''Varioscan – Erik'''

'''Casy-Zellzähler'''

'''TFA'''

== '''Unfälle''' ==
Beim ersten Aufenthalt im Labor mit den Schutzeinrichtungen (Notfallduschen, Augenduschen, Feuerlöscher, Sicherheitsschalter), Notfallnummern (Am Telefon neben dem Aufzug), Erste Hilfe Kasten (ebenfalls am Telefon neben dem Aufzug) und Fluchtwegen vertraut machen.

Bei Kontakt der Augen mit Chemikalien/Krankheitserregern: Sofort Ausspülen der Augen mit Hilfe der Augenduschen, dann die Ersthelfer (Tel.-Nr. '''2222''') benachrichtigen lassen.

Bei Kontakt der Haut/Laborkleidung mit Chemikalien, bei Brand der Laborkleidung:

Notfallduschen im Gang oder in Labor nutzen

Verletzungen/Schnitte: Ersthelfer kontaktieren

Bei Ausschütten von Lösungen/Krankheitserregern: sofort Unfallstelle kennzeichnen und absichern, dann erst mit Inaktivierung/Dekontamination beginnen

Feuer: Fluchtwege und Sammelpunkt einprägen

== '''Zentrifugen''' ==
Die Bedienung der Zentrifugen darf erst nach Einweisung durch die für Zentrifugen verantwortliche Person erfolgen (Susanne Feindler-Boeckh). Immer Zentrifugengefäße austarieren, bei vielen Zentrifugen stehen entsprechend befüllte Tara-Röhrchen bereits bereit. Nicht alle Zentrifugengefäße passen in jeden Rotor. Bitte auf richtige Passung achten und nie Gewalt anwenden, um Zentrifugenbecher in den Rotor zu laden. Ultrazentrifugenrotoren immer mit ALLEN Gehängen in der vorgesehenen Position betreiben. Bei der Zentrifugation von Suspensionen humanpathogener Bakterien sind die besonderen Betriebsanweisungen zu beachten.

== '''Ethidiumbromid''' ==
DNA-Färbung von Agarosegelen ausschließlich in der Dunkelkammer in dem dafür vorgesehen Färbe/Entfärbebad durchführen. Dort stehen auch Nitrilhandschuhe bereit, die nur für diesen Zweck und NICHT außerhalb der Dunkelkammer verwendet werden. EINE behandschuhte Hand reicht aus, so dass Ethidiumbromid-freie Gegenstände (z.B. Hauptschalter der Steckdose) mit der Handschuh-freien Hand bedient werden können.

Wasser für das Entfärbebad ist regelmäßig zu erneuern (mindestens täglich). Die Entfärbelösung kann ins Waschbecken entleert werden und durch normales VE-Wasser aufgefüllt werden.

== '''Gefahrstoffe''' ==
Bei der Arbeit mit Gefahrstoffen sind die gültigen Sicherheitsbestimmungen einzuhalten. Insbesondere gilt bei der Arbeit mit:

''Paraformaldehyd'': ist im ungelösten Zustand unter dem Abzug abzuwiegen.

''Mercaptoethanol-Lösungen'': sind nur unter dem Abzug zu handhaben.

''Brennbare und leicht flüchtige Substanzen (Methanol, Ethanol)'': Die Vorräte sind im Sicherheitsschrank aufzubewahren. Geringe Mengen (Tagesbedarf) können am Arbeitsplatz in gekennzeichneten Gefäßen aufbewahrt werden.

''Phenol und Phenolhaltige-Lösungen'': dürfen nur unter Abzug II gehandhabt werden. Dort sind auch Abfallbehälter für die Entsorgung von flüssigen bzw. festen/brennbaren Phenolhaltigen Abfällen.

Acrylamid

== '''Abfälle''' ==
Abfälle von giftigen Chemikalien, Farbstoffen, Fixantien, werden separat in beschrifteten Abfallkanistern und –flaschen gesammelt, bevor sie sachgemäß über das Chemiklaien-Abfalllager entsorgt werden.

== '''Betriebsärztliche Untersuchung''' ==
Jeder Mitarbeiter muss 1x/Jahr vom Betriebsarzt untersucht werden. Dazu wird bei Einstellung/Arbeitsbeginn ein Termin über Petra Schnurr organisiert. Die weiteren jährlichen Untersuchungen sind selbständig zu organisieren und eine Kopie der Unbedenklichkeitserklärung des Betriebsarztes ist nach erfolgter Untersuchung im Sekretariat bei Petra Schnurr abzugeben.

== '''Sicherheitsbelehrung''' ==
Eine allg. Sicherheitsbelehrung zur biologischen Sicherheit führt Stefan Kreft, Beauftragter für Biol. Sicherheit der Universität, 1-mal jährlich durch. Im Zusammenhang mit dem VTK gibt es zusätzlich 1-mal jährlich eine arbeitsplatzspezifische Sicherheitsbelehrung. Beide Veranstaltungen sind obligatorisch. Bei Arbeitsbeginn ist ebenfalls eine Sicherheitsbelehrung und Laboreinweisung zu absolvieren.

All About Safety

2025-06-05T11:58:53Z

ESO wikiadmin:

This Page is also available in [[Laborsicherheit|german]] 🇩🇪.

== Protective Clothing ==
In the laboratories, it is ALWAYS mandatory to wear a closed lab coat and safety goggles during all procedures. Special caution is required when handling liquid polyacrylamide (casting SDS-PAGE gels) and when working with acids or bases. In such cases, gloves must be worn in addition to the lab coat and goggles.

== Gas ==
Lab gas is centrally stored in Room ML in a safety cabinet. The gas supply should be turned off in the evening using the key switch. After completing gas-related work at the lab benches, the gas shut-off valve must be closed.

== Sharp Objects ==

* Needles/Razor Blades/Scalpels Exercise caution when working with these tools due to injury risk. Do NOT re-cap used needles—dispose of them immediately. These items must be discarded in clearly labeled yellow plastic containers (single-use). One container is located in the cold room, another under Fume Hood II.
* Pasteur Pipettes’’’ Pasteur pipettes should be collected in large, yellow plastic containers (reusable), labeled “Nur Pasteurpipetten / Pasteur Pipettes ONLY”. One container is located in each cell culture lab and one in the cold room. Please DO NOT dispose of other sharp items (scalpels, razor blades, needles, etc.) in these containers, as the contents will be autoclaved and then treated as glass waste.
* Glass Bottles/Broken Glass’’’ Broken glass and empty lab glass bottles should be disposed of in the designated containers.

== Training by Lab Supervisors ==
Cell Culture – Any new member must pass the introduction to the cell culture laboratory by SSJ before any work is carried out there. Further information here: [[S1 - Cell culture guidelines|'''S1 - Cell culture guidelines''']]

Fluorescence Microscope: xxx

Working in the S2 Lab (Infection Lab)

xxx

Working at the RNA Workstation

xxx

Microscopy – Marleen

Varioscan – Erik

Casy Cell Counter

TFA

== Accidents ==
On your first visit to the lab, familiarize yourself with the emergency equipment (emergency showers, eye wash stations, fire extinguishers, safety switches), emergency numbers (on the phone next to the elevator), first aid kit (also next to the elevator), and escape routes.

In case of eye contact with chemicals/pathogens: Immediately rinse eyes using the eye wash station, then notify a first responder (Phone number: ‘’‘2222’’’).

In case of skin/lab coat contact with chemicals, or clothing fire:

Use the emergency showers in the hallway or lab.

For injuries or cuts: Contact a first responder.

In case of spilled solutions or pathogens: Mark and secure the accident site immediately, then begin decontamination or inactivation.

Fire: Memorize escape routes and the designated assembly point.

== Centrifuges ==
Centrifuges may only be operated after instruction by the responsible person (Susanne Feindler-Boeckh). Always balance centrifuge tubes—pre-filled balancing tubes are provided with many centrifuges. Not all tubes are compatible with all rotors. Ensure proper fitting and NEVER force containers into the rotor. Ultracentrifuge rotors must always be operated with ALL buckets in place and properly positioned. Special safety instructions apply when centrifuging suspensions of human-pathogenic bacteria.

== Ethidium Bromide ==
DNA staining of agarose gels must be done exclusively in the darkroom using the designated staining/destaining baths. Nitrile gloves are provided there for this specific purpose and MUST NOT be used outside the darkroom. One gloved hand is sufficient, allowing the ungloved hand to operate clean objects (e.g., main power switch).

The water in the destaining bath must be replaced regularly (at least daily). The used solution can be poured into the sink and replaced with deionized water (DI water).

== Hazardous Substances ==
Current safety regulations must be followed when handling hazardous substances. Specific precautions include:

''Paraformaldehyde'': Must be weighed under the fume hood while in solid form.

''Mercaptoethanol solutions'': Must only be handled under the fume hood.

''Flammable and volatile substances (methanol, ethanol)'': Stocks must be stored in a safety cabinet. Small quantities (daily use) may be kept in labeled containers at the bench.

''Phenol and phenol-containing solutions'': Must only be handled under Fume Hood II. Special waste containers for liquid and solid/flammable phenol waste are also located there.

''Acrylamide'': [Consider adding handling instructions]

== Waste Disposal ==
Waste from toxic chemicals, dyes, and fixatives must be collected separately in labeled containers (canisters and bottles) before being properly disposed of via the chemical waste facility.

== Occupational Health Examinations ==
Each employee must undergo a medical examination by the company physician once per year. An initial appointment is arranged through Petra Schnurr when employment begins. All subsequent annual appointments must be scheduled independently. A copy of the fitness certificate must be submitted to the secretariat (Petra Schnurr) after the examination.

== Safety Instructions ==
General safety training on biological safety is conducted once a year by Stefan Kreft, the university’s biological safety officer. Additional workplace-specific safety instruction is held once a year in connection with the VTK. Both are mandatory. A safety briefing and lab orientation must also be completed at the start of employment.

All About Safety

2025-06-05T11:58:33Z

ESO wikiadmin:

This Page is also available in [[Laborsicherheit|german]] 🇩🇪.

== Protective Clothing ==
In the laboratories, it is ALWAYS mandatory to wear a closed lab coat and safety goggles during all procedures. Special caution is required when handling liquid polyacrylamide (casting SDS-PAGE gels) and when working with acids or bases. In such cases, gloves must be worn in addition to the lab coat and goggles.

== Gas ==
Lab gas is centrally stored in Room ML in a safety cabinet. The gas supply should be turned off in the evening using the key switch. After completing gas-related work at the lab benches, the gas shut-off valve must be closed.

== Sharp Objects ==

* Needles/Razor Blades/Scalpels Exercise caution when working with these tools due to injury risk. Do NOT re-cap used needles—dispose of them immediately. These items must be discarded in clearly labeled yellow plastic containers (single-use). One container is located in the cold room, another under Fume Hood II.
* Pasteur Pipettes’’’ Pasteur pipettes should be collected in large, yellow plastic containers (reusable), labeled “Nur Pasteurpipetten / Pasteur Pipettes ONLY”. One container is located in each cell culture lab and one in the cold room. Please DO NOT dispose of other sharp items (scalpels, razor blades, needles, etc.) in these containers, as the contents will be autoclaved and then treated as glass waste.
* Glass Bottles/Broken Glass’’’ Broken glass and empty lab glass bottles should be disposed of in the designated containers.

== Training by Lab Supervisors ==
Cell Culture – Any new member must pass the introduction to the cell culture laboratory by SSJ before any work is carried out there. Weitere Informationen unter [[S1 - Cell culture guidelines|'''S1 - Cell culture guidelines''']]

Fluorescence Microscope: xxx

Working in the S2 Lab (Infection Lab)

xxx

Working at the RNA Workstation

xxx

Microscopy – Marleen

Varioscan – Erik

Casy Cell Counter

TFA

== Accidents ==
On your first visit to the lab, familiarize yourself with the emergency equipment (emergency showers, eye wash stations, fire extinguishers, safety switches), emergency numbers (on the phone next to the elevator), first aid kit (also next to the elevator), and escape routes.

In case of eye contact with chemicals/pathogens: Immediately rinse eyes using the eye wash station, then notify a first responder (Phone number: ‘’‘2222’’’).

In case of skin/lab coat contact with chemicals, or clothing fire:

Use the emergency showers in the hallway or lab.

For injuries or cuts: Contact a first responder.

In case of spilled solutions or pathogens: Mark and secure the accident site immediately, then begin decontamination or inactivation.

Fire: Memorize escape routes and the designated assembly point.

== Centrifuges ==
Centrifuges may only be operated after instruction by the responsible person (Susanne Feindler-Boeckh). Always balance centrifuge tubes—pre-filled balancing tubes are provided with many centrifuges. Not all tubes are compatible with all rotors. Ensure proper fitting and NEVER force containers into the rotor. Ultracentrifuge rotors must always be operated with ALL buckets in place and properly positioned. Special safety instructions apply when centrifuging suspensions of human-pathogenic bacteria.

== Ethidium Bromide ==
DNA staining of agarose gels must be done exclusively in the darkroom using the designated staining/destaining baths. Nitrile gloves are provided there for this specific purpose and MUST NOT be used outside the darkroom. One gloved hand is sufficient, allowing the ungloved hand to operate clean objects (e.g., main power switch).

The water in the destaining bath must be replaced regularly (at least daily). The used solution can be poured into the sink and replaced with deionized water (DI water).

== Hazardous Substances ==
Current safety regulations must be followed when handling hazardous substances. Specific precautions include:

''Paraformaldehyde'': Must be weighed under the fume hood while in solid form.

''Mercaptoethanol solutions'': Must only be handled under the fume hood.

''Flammable and volatile substances (methanol, ethanol)'': Stocks must be stored in a safety cabinet. Small quantities (daily use) may be kept in labeled containers at the bench.

''Phenol and phenol-containing solutions'': Must only be handled under Fume Hood II. Special waste containers for liquid and solid/flammable phenol waste are also located there.

''Acrylamide'': [Consider adding handling instructions]

== Waste Disposal ==
Waste from toxic chemicals, dyes, and fixatives must be collected separately in labeled containers (canisters and bottles) before being properly disposed of via the chemical waste facility.

== Occupational Health Examinations ==
Each employee must undergo a medical examination by the company physician once per year. An initial appointment is arranged through Petra Schnurr when employment begins. All subsequent annual appointments must be scheduled independently. A copy of the fitness certificate must be submitted to the secretariat (Petra Schnurr) after the examination.

== Safety Instructions ==
General safety training on biological safety is conducted once a year by Stefan Kreft, the university’s biological safety officer. Additional workplace-specific safety instruction is held once a year in connection with the VTK. Both are mandatory. A safety briefing and lab orientation must also be completed at the start of employment.