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5 June 2025

  • 14:2714:27, 5 June 2025 Protein expression and purification (hist | edit) [7,148 bytes] ESO wikiadmin (talk | contribs) (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...") Tag: Visual edit
  • 14:1714:17, 5 June 2025 Testexpression in Pichia pastoris (hist | edit) [1,506 bytes] ESO wikiadmin (talk | contribs) (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...") Tag: Visual edit
  • 14:0714:07, 5 June 2025 Transformation of Pichia pastoris (hist | edit) [1,499 bytes] ESO wikiadmin (talk | contribs) (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...") Tag: Visual edit
  • 14:0414:04, 5 June 2025 Generation of competent Pichia pastoris (hist | edit) [849 bytes] ESO wikiadmin (talk | contribs) (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...") Tag: Visual edit
  • 13:5613:56, 5 June 2025 Purification of GST-fusion proteins (hist | edit) [2,329 bytes] ESO wikiadmin (talk | contribs) (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...") Tag: Visual edit
  • 13:5313:53, 5 June 2025 Expression of GST-fusion proteins (hist | edit) [2,003 bytes] ESO wikiadmin (talk | contribs) (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...") Tag: Visual edit
  • 13:5013:50, 5 June 2025 Bacterial Pulldown with soluble receptors (hist | edit) [1,616 bytes] ESO wikiadmin (talk | contribs) (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<br>Cell culture supernatants (e.g. OptiMEM) harvested from 293cells transiently transfected with constructs encoding soluble CEACAM- ectodomains fused to GFP<br>Polyclonal rabbit anti-GFP antibody (AG Hauck)<br>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}}...") Tag: Visual edit
  • 13:4813:48, 5 June 2025 Opsonizing bacteria with Fc fusion proteins (hist | edit) [599 bytes] ESO wikiadmin (talk | contribs) (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...") Tag: Visual edit
  • 12:4312:43, 5 June 2025 Immunoprecipitation (hist | edit) [2,125 bytes] ESO wikiadmin (talk | contribs) (Created page with "This is an original excerpt from MHE's digital labbook from 25 April 2025. {{Protokoll-Box}} = Immunoprecipitation = -       THP-1 Cells (WT, PPM1F KO (clone D7), PPM1F KO + PPM1F WT (clone C9), PPM1F KO+ PPM1F D360A (clone A5)) were count and 3x10<sup>6</sup> cells were lysed using 1000 µl RIPA lysis buffer (10 µg/ml Aprotinin,10 µM Benzamidin, 5 µg/ml Leupepin, 1 µM PMSF, 10 nM pNPP, 2 µM cytochalasinD, cpd26, 1x Phosstop) -       Cells were vortexted, s...") Tag: Visual edit
  • 12:2812:28, 5 June 2025 Bio-Rad GelDoc (hist | edit) [4,137 bytes] ESO wikiadmin (talk | contribs) (Created page with "= Bio-Rad GelDoc XR+ System - Overview = == What is the GelDoc XR+ System? == The GelDoc XR+ System from Bio-Rad is a reliable gel documentation system designed for high-quality imaging of nucleic acid and protein gels. This system combines proven CCD camera technology with versatile illumination options to provide consistent, publication-quality images for routine gel documentation needs. == Main Applications == === Nucleic Acid Gel Documentation === * '''Agarose ge...") Tag: Visual edit
  • 12:2412:24, 5 June 2025 Bio-Rad ChemiDoc Touch (hist | edit) [2,807 bytes] ESO wikiadmin (talk | contribs) (Created page with "= Bio-Rad ChemiDoc Touch System - Overview = == What is the ChemiDoc Touch System? == The ChemiDoc Touch System from Bio-Rad is a versatile imaging system for documentation and analysis of gels and blots. It combines high-resolution CCD camera technology with intuitive touchscreen operation and enables capture of fluorescence, chemiluminescence, and visible light images. == Main Applications == === Gel Documentation === * '''Agarose gels:''' DNA/RNA electrophoresis w...") Tag: Visual edit
  • 12:1712:17, 5 June 2025 Thermo Scientific Varioskan Flash eng (hist | edit) [4,117 bytes] ESO wikiadmin (talk | contribs) (Created page with "This page is also available in german 🇩🇪. = Varioskan Flash - Basic Operation Guide = == Overview == The Varioskan Flash is a multimode microplate reader for various detection methods (absorbance, fluorescence, luminescence, time-resolved fluorescence). This guide provides a basic overview - '''the manual remains essential for detailed protocols!''' == Preparation == === 1. Power on the instrument === * Activate main power...") Tag: Visual edit
  • 12:1112:11, 5 June 2025 Thermo Scientific Varioskan Flash (hist | edit) [4,438 bytes] ESO wikiadmin (talk | contribs) (Created page with "Diese Seite ist auch auf Englisch 🇬🇧 verfügbar. = Varioskan Flash - Grundlegende Bedienung = == Überblick == Der Varioskan Flash ist ein Multimode-Mikroplatten-Reader für verschiedene Detektionsmethoden (Absorbanz, Fluoreszenz, Lumineszenz, Zeit-aufgelöste Fluoreszenz). Diese Anleitung bietet einen ersten Überblick - '''das Handbuch bleibt unverzichtbar für detaillierte Protokolle!''' == Vorbereitung == === 1. Ger...") Tag: Visual edit

3 June 2025

  • 16:2216:22, 3 June 2025 S1 - Cell culture guidelines (hist | edit) [2,363 bytes] ESO wikiadmin (talk | contribs) (Created page with "'''Getting started:''' * Turn on hood, open bench  needs several minutes, wait for green light * Tie back hair, push back lose sleeves; disinfect hands & arms, put on gloves (can be reused) * Wipe down bench and window with 70% EtOH * Gather all material you need, spray with 70% EtOH and wipe down before placing under hood * Arrange items under hood towards the back of the bench ⇒Keep in mind that you should work a minimum of 15cm inward from the outside edge of th...") Tag: Visual edit
  • 13:0313:03, 3 June 2025 Laborsicherheit (hist | edit) [6,165 bytes] ESO wikiadmin (talk | contribs) (Created page with "== '''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...") Tag: Visual edit

2 June 2025

  • 09:2809:28, 2 June 2025 FBA-staining of bacteria (hist | edit) [1,326 bytes] ESO wikiadmin (talk | contribs) (Created page with "{{Protokoll-Mix|Inhalt=(5-(6)-carboxyfluorescein succinimidyl ester (CFSE, FAM SE, Fluorescein)<br>NHS-Biotin<br>PBS++ (PBS, 45 nM CaCl 2, 35 nM MgCl 2)<br>4% Paraformaldehyde (PFA)<br>Blocking solution (10% FCS in PBS ++)<br>PBS<br>Streptavidin-AlexaFluor647}} == Procedure == * Dilute 0.2 mg NHS-Biotin in 1 ml PBS (prewarm Biotin to RT before opening the tube) * Collect bacteria from plate in PBS, centrifuge for 4 min, 4000 rpm, and resuspend the pellet in 700 l PB...") Tag: Visual edit
  • 09:0409:04, 2 June 2025 Gentamicin Protection Assay (hist | edit) [1,691 bytes] ESO wikiadmin (talk | contribs) (Created page with "== Procedure == * Aspirate cell culture medium with a pasteur pipette * Rince attaching cells carefully with cold PBS, aspirate PBS * Add 1.5 mL Trypsin/EDTA and study the detachment of the cells in the microscope * Inactivate Trypsin/EDTA with 3.5 mL medium, transfer the cell suspension into a 15 mL centrifuge tube * Pipette 20 mL of the cell suspension into a Neubauer counting chamber * Centrifuge the remaining cells at 600 rpm for 3 min * During centrifugation: Deter...") Tag: Visual edit
  • 08:5308:53, 2 June 2025 Monitoring bacterial growth (hist | edit) [2,520 bytes] ESO wikiadmin (talk | contribs) (Created page with "{{Protokoll-Mix|Inhalt=Depending on the bacterium you want to analyse, you should determine beforehand: *Growth temperature *Shaking intensity *Generation time *Appropriate medium *Liquid or plate culture}} == Pre-culture == Streak out bacteria one to two days prior to the experiment: two days if you want to do a selection for single clones or if you have to incubate them on antibiotics. Addition of antibiotics usually hampers with the growth - for the determination o...") Tag: Visual edit
  • 08:4908:49, 2 June 2025 Transformation of Neisseria gonorrhoeae (hist | edit) [1,000 bytes] ESO wikiadmin (talk | contribs) (Created page with "{{Protokoll-Mix|Inhalt=Gonococcal strain which is Opa-postive /Pili positive PPM medium (15g proteose pepton, 5g NaCl, 4g KH 2PO4 1g K2HPO4, 1g soluble starch, ad 1l H20 ph: 7.5) + vitamin mix cloned and sequenced analysed gonococcal DNA which contains the spectinomycin resistance cassette and the Neisserial DNA uptake sequence (DUS) (plasmid DNA}} == Procedure == * Ngo strain (O+/P+) were grown overnight (37°C) on GC agar plates in a humid atmosphere with 5% CO 2 * N...") Tag: Visual edit
  • 08:3908:39, 2 June 2025 Transformation of plasmids in E. coli (hist | edit) [1,266 bytes] ESO wikiadmin (talk | contribs) (Created page with " == Procedure == * Before transformation, appropriate aliquots of competent bacteria should be thawn on ice. * Add the plasmid DNA to 100 μl competent E. coli in a snap-cap tube and mix well. * Incubate the samples 30 min on ice. * Transfer samples for 75 seconds to a 42°C water bath or heating block and quickly back on ice. * Add 1 ml LB-Medium and incubate the bacteria for 1h at 37°C (220 rpm). * Transfer the bacterial suspension from the snap-cap tube to Eppendorf...") Tag: Visual edit

30 May 2025

29 May 2025

  • 18:1218:12, 29 May 2025 PCR approach in 50 μL with hot-start and/or touch-down (preparative PCR) (hist | edit) [1,801 bytes] ESO wikiadmin (talk | contribs) (Created page with "{{Protokoll-Mix|Inhalt=20 mM dNTPs 10x Taq/Pfu buffer 2 Primers, 10 μM DNA template, 100 – 200 ng/μL DNA polymerases (Pfu, Taq)}} == Procedure == As usually more than one PCR sample is run simultaneously, reaction and enzyme mix are prepared separately. The enzyme mix is added to the reaction mix after starting the PCR and after pre-heating the reaction mix to 94 ºC (hot start). A typical 50 μL approach that yields enough material for later cloning procedures...") Tag: Visual edit
  • 18:0418:04, 29 May 2025 Design principles for PCR primers used for amplification and cloning of cDNA (hist | edit) [2,490 bytes] ESO wikiadmin (talk | contribs) (Created page with "'''Restriction Enzyme Sites:''' For conventional cloning with restriction enzymes, the recognition sites are added to the 5’ end of the primer. Check that the involved '''restriction enzymes do NOT cut within the cloned cDNA''' and that they do '''NOT cut the vector outside the intended position'''. Make sure to preserve the reading frame, if the DNA is cloned 3’ or 5’ of existing ORFs (e.g. GFP) in the vector. Add 3 to 4 meaningless nucleotides (e.g. ATAA-) at the...") Tag: Visual edit
  • 17:5617:56, 29 May 2025 Agarose gel electrophoresis (hist | edit) [1,341 bytes] ESO wikiadmin (talk | contribs) (Created page with "{{Protokoll-Mix |titel=PCR Reaction mix (Pipett scheme) |TAE buffer (1x) 4.84 g Tris-Base, 1 mM EDTA, 1.14 mL acetic acid, fill 1 L with A.bidest. Agarose }}")
  • 16:1816:18, 29 May 2025 Vorlage:Homepage-Grid (hist | edit) [1,174 bytes] ESO wikiadmin (talk | contribs) (Created page with "<div style="display: grid; grid-template-columns: 1fr 1fr; gap: 20px; margin: 20px 0;"> <div style="grid-column: 1;"> {{Colored-Box |title={{{title1|Titel 1}}} |content={{{content1|Inhalt Box 1}}} |background={{{bg1|#e8f4fd}}} |border={{{border1|#3366cc}}} |link={{{link1|}}} |link-text={{{linktext1|Mehr →}}} }} </div> <div style="grid-column: 2;"> {{Colored-Box |title={{{title2|Titel 2}}} |content={{{content2|Inhalt Box...")
  • 16:1816:18, 29 May 2025 Vorlage:Colored-Box (hist | edit) [707 bytes] ESO wikiadmin (talk | contribs) (Created page with "<div class="colored-box" style="background: {{{background|#f8f9fa}}}; border: 1px solid {{{border|#a2a9b1}}}; border-radius: 4px; padding: 16px; margin: 10px 0; box-shadow: 0 1px 3px rgba(0,0,0,0.1);"> {{#if:{{{title|}}}|<h3 style="margin-top: 0; color: {{{title-color|#0645ad}}}; border-bottom: 1px solid {{{border|#a2a9b1}}}; padding-bottom: 8px; margin-bottom: 12px;">{{{title}}}</h3>}} <div style="color: {{{text-color|#333}}};"> {{{content|Box-Inhalt hier}}} <...")
  • 16:1516:15, 29 May 2025 Vorlage:Tip (hist | edit) [170 bytes] ESO wikiadmin (talk | contribs) (Created page with "<div style="border-left: 4px solid #8b5cf6; background: #f5f3ff; padding: 12px; margin: 16px 0; border-radius: 2px;"> <strong>💡 Tipp:</strong> {{{1|Tipp-Text}}} </div>")
  • 16:1416:14, 29 May 2025 Vorlage:Info (hist | edit) [179 bytes] ESO wikiadmin (talk | contribs) (Created page with "<div style="border-left: 4px solid #00af89; background: #f0fdf4; padding: 12px; margin: 16px 0; border-radius: 2px;"> <strong>ℹ️ Info:</strong> {{{1|Informationstext}}} </div>")
  • 16:1416:14, 29 May 2025 Vorlage:Warning (hist | edit) [174 bytes] ESO wikiadmin (talk | contribs) (Created page with "<div style="border-left: 4px solid #d73027; background: #fdf2f2; padding: 12px; margin: 16px 0; border-radius: 2px;"> <strong>⚠️ Warning:</strong> {{{1|Warntext}}} </div>")
  • 16:0916:09, 29 May 2025 Vorlage:Note (hist | edit) [174 bytes] ESO wikiadmin (talk | contribs) (Hinweisbox)
  • 15:4415:44, 29 May 2025 Vorlage:Print-Button (hist | edit) [1,004 bytes] ESO wikiadmin (talk | contribs) (Print/Export-Button-Vorlage erstellt)
  • 15:1015:10, 29 May 2025 Colony PCR (hist | edit) [1,640 bytes] ESO wikiadmin (talk | contribs) (Created page with "Reaction mix: H<sub>2</sub>O                                         16.5 μL 10x Taq/Pfu buffer                       2 μL Primer 1                                    0.4 μL (10 pmol) Primer 2                                    0.4 μL (10 pmol) dNTP-Mix                                  0.2 μL Taq polymerase                        0.5 μL total              ...") Tag: Visual edit

21 May 2025

  • 14:1414:14, 21 May 2025 Restriction digest of plasmid DNA (hist | edit) [1,054 bytes] ESO wikiadmin (talk | contribs) (Created page with "{| class="wikitable" |+ !Materials |- |Restriction enzyme stock (–20°C) |- |Appropriate 10x enzyme buffer (–20°C) |- |Sterile dd H<sub>2</sub>O |- |Depending on the digestion '''(optional)''': '''''BSA (bovine serum albumin)''''': To ensure 100% activity of the enzymes, 100 µg/ml BSA should be added (stored at –20°C) |} == Procedure for analysis purposes == * 300 ng of DNA samples (e.g. control digest of plasmid minipreps) * 1 µl appropriate enzyme buffer (1...") Tag: Visual edit
  • 14:1114:11, 21 May 2025 Plasmid-Miniprep (Birnboim – Dooley protocol) (hist | edit) [1,327 bytes] ESO wikiadmin (talk | contribs) (Created page with "{| class="wikitable" |+ !Materials |- |Buffer P1: Solve 6.055 g Tris Base and 3.722g EDTA x 2H<sub>2</sub>O in 800 ml H<sub>2</sub>0. 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 H<sub>2</sub>0. Add 50 ml 20% SDS. Store at RT. |- |Puffer P3: 294.45g KAc in 500 ml H<sub>2</sub>O. Adjust pH to 5.5 with glacial acetic acid (about 110 ml acid needed), fill up to 1L with H<sub>...") Tag: Visual edit

20 May 2025

  • 15:2715:27, 20 May 2025 Vorlage Klonierungsprojekt (hist | edit) [2,298 bytes] ESO wikiadmin (talk | contribs) (Created page with "<noinclude> == Vorlage für Klonierungsprojekte == Diese Seite dient als Vorlage für neue gentechnische Arbeiten im Labor (z. B. PCR-Klonierungen). Kopiere den folgenden Inhalt in eine neue Wiki-Seite und passe ihn projektbezogen an. '''Hinweis:''' Alle Datumsangaben, Enzyme, Vektoren etc. bitte entsprechend aktualisieren.</noinclude> == Projekttitel == ''(z. B. Klonierung von GFP-tagged PAK1 in pcDNA3.1)'' * Startdatum: __.__.20__ * Bearbeiter: Benutzer:DeinN...") Tag: Visual edit
  • 15:1615:16, 20 May 2025 Flow chart of a PCR-based cloning project (hist | edit) [3,199 bytes] ESO wikiadmin (talk | contribs) (Created page with "== Experimenteller Ablaufplan eines klassischen PCR-basierten Klonierungsprojekts == ''(LIC-basiertes Klonieren kann in vergleichbarer Zeit mit ähnlichen Schritten durchgeführt werden)'' === Montag === * PCR (5 × 50 µl Reaktionen im Temperaturgradienten) * 5 µl jeder PCR-Reaktion auf Agarosegel laden, Produktgröße & Qualität kontrollieren * 2–3 Proben kombinieren (3 × 45 µl), 5 µl DpnI hinzufügen, 1 h bei 37 °C inkubieren * 5-faches Volumen Puffer PB (...") Tag: Visual edit: Switched
  • 15:0915:09, 20 May 2025 Fluorescent Staining of Cryosections (hist | edit) [881 bytes] ESO wikiadmin (talk | contribs) (Created page with "{| class="wikitable" |+ !Materials |- |PBS pH 7.4 |- |Blocking buffer (1% BSA, 1% serum and 0.1% Triton X-100 in PBS) |- |Corresponding 1st and 2nd antibodies |- |Barrier pen |- |Mounting medium |} === Procedure: === * Thaw the slides at room temperature for 10-20 min * The slides are washed for 10 min in PBS * Block non-specific staining by incubating samples in blocking buffer for 30 minutes at RT * Surround the tissue with a hydrophobic barrier using a barrier pen...") Tag: Visual edit
  • 15:0615:06, 20 May 2025 Tissue/embryo preparation fo cryosectioning (hist | edit) [1,081 bytes] ESO wikiadmin (talk | contribs) (Created page with "{| class="wikitable" |+ !Materials |- |PBS pH 7.4 |- |4% PFA in PBS |- |10%, 20% and 30% sucrose solution in PBS |- |OCT Tissue-Tek |- |Histology moulds |- |Pre-coated glass slides |- |Dry ice |} === Procedure: === * Tissues/ anesthetized embryos are dissected and placed into 4% PFA solution overnight at 4° C * The next day the specimen is washed with PBS and transferred to a 10% sucrose solution for 3 h at 4° C (keep at 4°C until specimen sinks) * The sample is tra...") Tag: Visual edit
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