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A user with 403 edits. Account created on 13 February 2025.
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20 May 2025

  • 15:2015:20, 20 May 2025 diff hist +60 Methods and Protocols No edit summary Tag: Visual edit
  • 15:1615:16, 20 May 2025 diff hist +3,152 N Flow chart of a PCR-based cloning project 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
  • 15:1515:15, 20 May 2025 diff hist +3 Methods and Protocols →Molecular Biology Tag: Visual edit
  • 15:0915:09, 20 May 2025 diff hist +881 N Fluorescent Staining of Cryosections 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..." current Tag: Visual edit
  • 15:0615:06, 20 May 2025 diff hist +1,081 N Tissue/embryo preparation fo cryosectioning 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..." current Tag: Visual edit
  • 15:0315:03, 20 May 2025 diff hist +1,704 N Bacterial invasion assay by flow cytometry Created page with "{| class="wikitable" |+ !Materials (for 6 cm dishes) |- |CFSE |- |FACS buffer (1% (F)CS in PBS, 50-100 mL (ice-cold) |- |Trypsin/EDTA (1mL/dish) |- |Medium with serum according to cell type (3mL/dish) |- |15 mL PP falcon tubes |- |Trypan blue solution, 0.4 mg/mL |- |Eppendorf tubes 1.5mL (1 per dish) |} === Procedure === Bacteria: * 2 days before the experiment, streak bacteria from glycerol stock on selective GC or TSB agar plates (with antibiotics when needed) and in..." current Tag: Visual edit
  • 15:0015:00, 20 May 2025 diff hist −97 Staining of cells for flow cytometry No edit summary current Tag: Visual edit
  • 09:5709:57, 20 May 2025 diff hist +1,760 N Staining of cells for flow cytometry Created page with "{| class="wikitable" |+ ! ! ! ! |- |FACS buffer: |1xPBS FCS (heat-inactivated, final concentration: 5%) 5% Natriumazid (final concentration: 0.1%) | | |- |1st antibody: |primary antibody (approx. dilution 1:200) | | |- |2nd antibody: |secondary antibody (approx. dilution 1:500) fluorescence- labelled | | |- |controls: |sample with irrelevant primary antibody and secondary antibody sample with secondary antibody only if possible untransfected cells stained with both antib..." Tag: Visual edit
  • 09:5409:54, 20 May 2025 diff hist +2,243 N Scanning electron microscopy (SEM) Created page with "{| class="wikitable" |+ !Materials |- |'''Fixation buffer''': 3% Formaldehyde, 2% Glutardialdehyde, 10 mM CaCl<sub>2</sub>, 10 mM MgCl<sub>2</sub>, 90 mM Sucrose in 100 mM HEPES, pH 7.2 |- |'''Washing Buffer''': 10 mM CaCl<sub>2</sub>, 10 mM MgCl<sub>2</sub>, 90 mM Sucrose in 100 mM HEPES, pH 7.2 |- |'''HEPES buffer:''' 100 mM, pH 7.2 |- |'''Graded ethanol series:''' 30%, 50%, 70%, 90%, 100% ethanol p.a. |} === Procedure: === '''1A. ''Organs''''' of infected animals are..." current Tag: Visual edit
  • 09:4709:47, 20 May 2025 diff hist −8 OPTIC (Opa protein triggered integrin clustering) No edit summary current Tag: Visual edit
  • 09:4709:47, 20 May 2025 diff hist −83 OPTIC (Opa protein triggered integrin clustering) No edit summary Tag: Visual edit
  • 09:4309:43, 20 May 2025 diff hist +2,679 N OPTIC (Opa protein triggered integrin clustering) Created page with "{| class="wikitable" |+ !Materials |- |1x PBS |- |Trypsin/EDTA |- |GC-Agar plates with CAM/ERM and without antibiotics Coverslips (round, 12 mm) |- |24 well plate |- |HEK culture medium (DMEM + 10% CS) Suspension medium (DMEM + 0.25% BSA) 4% paraformaldehyde (PFA) in PBS |- |Permeabilization solution (0.4% Triton-X-100 in PBS) Blocking solution (10% heat inactivated CS in PBS) Mounting medium |} === Procedure === ==== '''Day1:''' ==== ==== Seed HEK293T cells 1:40 in 6..." Tag: Visual edit
  • 09:3909:39, 20 May 2025 diff hist +1,282 N Live cell microscopy Created page with "=== Preparation of culture dishes === * In 3.5 cm culture dishes (Sarstedt 83.1800 35x10 mm) a hole is drilled with 14 mm in diameter (will be done by the uni garage) * Before sticking the coverslips (Menzel 24 mm, Stärke 1,5; CB00240RAC20MNZ#0) to the dishes spray them with 70% ethanol and wipe with a KimWipe tissue * The coverslips are pretreated as following: ** Wash with a mixture of 50% v/v Aceton and 50% v/v EtOH ** Wash with 1xPBS ** Wash with MilliQ water ** Dr..." current Tag: Visual edit
  • 09:3609:36, 20 May 2025 diff hist −369 Immunofluorescence staining of focl adhesion proteins No edit summary current Tag: Visual edit

7 May 2025

7 March 2025

  • 10:4910:49, 7 March 2025 diff hist +1,846 N Immunofluorescence staining of focl adhesion proteins Created page with "Materials PBS++ (1x PBS + 0.9mM CaCl<sub>2</sub> + 0.5 mM MgCl<sub>2</sub>) Coverslips (round, 12 mm) 24 well plate 4% paraformaldehyde (PFA) in PBS Permeabilization/fixation solution (4% PFA/PBS + 0.1% Triton-X-100) Blocking solution (10% heat inactivated CS in PBS) Mounting medium {| class="wikitable" |+ !Materials ! |- | |PBS++ (1x PBS + 0.9mM CaCl<sub>2</sub> + 0.5 mM MgCl<sub>2</sub>) |- | |Coverslips (round, 12 mm) |- | |24 well plate |- | |4% paraformaldehyd..." Tag: Visual edit
  • 10:4710:47, 7 March 2025 diff hist +1,229 N Immunofluorescence staining Created page with "{| class="wikitable" |+ !Materials ! ! ! |- | |PBS++ (50 ml PBS; 25 μl 2.5M CaCl<sub>2</sub> ; 1M 50 μl MgCl<sub>2</sub>) | | |- | |Permeabilization solution (PBS++; 10% FCS; 0.2% Saponin; alternatively 0.2% | | |- | |Triton X-100 may be used instead of saponin as a detergent) | | |- | |Blocking solution (PBS++; 10% FCS) | | |- | |First and second antibody | | |- | |Mounting medium | | |- | |Nail polish | | |} === Procedure === ---- * After fixation wash (3 x with PB..." current Tag: Visual edit
  • 10:4510:45, 7 March 2025 diff hist +1,533 N Preparation of samples for confocal microscopy Created page with "{| class="wikitable" |+ !Materials ! |- | |Coverslips (round, 12 mm) |- | |24-well plate |- | |4% Paraformaldehyde (PFA) in PBS or |- | |ice-cold methanol or acetone (-20°C) in porcelain well plate |} === Procedure === ---- Cells: * One day before seeding the cells: distribute cleaned and sterilized coverslips in a 24-well plate and coat with gelatine-, poly-L-lysine (10 µg/mL), or fibronectin (2 µg/mL) solution in PBS overnight (o/n) at 4°C * Aspirate coating so..." current Tag: Visual edit
  • 10:4310:43, 7 March 2025 diff hist +1,796 N Direct labelling of antibodies with fluorescent dyes Created page with "{| class="wikitable" |+ !Materials ! |- | |Pacific Blue succinimidyl ester (absMAX= 416nm; emMAX= 451nm) |- | |5-(6)-carboxyfluorescein succinimidyl ester (FAM, SE) (absMAX= 494nm; emMAX= 518nm) |- | |5-(6)-carboxytetramethylrhodamine succinimidyl ester (TAMRA, SE) (absMAX= 555nm; emMAX= 580nm) |- | |AlexaFluor-647 succinimidyl ester (absMAX= 650nm; emMAX= 665nm) 1 x phosphate-buffered saline (PBS) |- | |1M sodium bicarbonate (pH 8.5) |- | |STOP buffer (prepare fresh): 1..." current Tag: Visual edit
  • 10:3910:39, 7 March 2025 diff hist +20 Methods and Protocols No edit summary Tag: Visual edit

27 February 2025

  • 14:5114:51, 27 February 2025 diff hist −54 All About Safety No edit summary Tag: Visual edit
  • 14:5014:50, 27 February 2025 diff hist −12 Lab Etiquette No edit summary Tag: Visual edit
  • 14:4914:49, 27 February 2025 diff hist −5,386 Lab Etiquette No edit summary Tag: Visual edit
  • 14:4414:44, 27 February 2025 diff hist +1,139 N Dot Blot Created page with "{| class="wikitable" |+ !Materials ! |- | |Nitrocellulose membrane / Whatman paper |- | |TBS-T |- | |Ponceau S |- | |Blocking Solution |- | |First and Second Antibody |- | |ECL solution and H<sub>2</sub>O<sub>2</sub> |} === Procedure === ---- * Whatman paper and membrane have to be wet  first whatman on dot blot, followed by membrane. Top of dot blot holder has to be firmly secured with screws on the lower part! * Apply 50 µl of probe with equivalent amount of prot..." current Tag: Visual edit
  • 14:4214:42, 27 February 2025 diff hist +87 Western Blot: stripping of the membrane No edit summary current Tag: Visual edit
  • 14:4014:40, 27 February 2025 diff hist +1,146 N Western Blot: stripping of the membrane Created page with "{| class="wikitable" |+ !Materials ! |- |Mild Stripping Buffer |15 g Glycin, 1 g SDS, 10 ml Tween20 in 1L ddH<sub>2</sub>O pH2.2 |- |Harsh Stripping Buffer |40 ml Stacking gel buffer (0,5M Tirs/HCl, pH6,8) 10 ml SDS 20%, 200 ml ddH<sub>2</sub>O |- | |1X PBS |- | |TBST |} == Procedure mild stripping == * Remove ECL from membrane by washing membrane briefly with ddH<sub>2</sub>O * Wash membrane 2x in Mild stripping buffer for 10 min * Wash membrane 2x in PBS for 10 min *..." Tag: Visual edit
  • 14:2914:29, 27 February 2025 diff hist +1,855 N Western Blot: probing of the membrane Created page with "{| class="wikitable" |+ !Materials ! |- |Staining Solution |250 mL isopropanol; 100 mL acetic acid; 650 mL H<sub>2</sub>O; 0.03% Coomassie |- |Destaining Solution |10% (v/v) isopropanol, 10% (v/v) acetic acid |- |Blocking Solution |TBS-T; 2% BSA; 0.05% NaN<sub>3</sub> |- |TBS-Tween |200 mL 10x TBS; 10 mL Tween; ad 2 L H<sub>2</sub>O |- | |ECL-Solution |- | |H<sub>2</sub>O<sub>2</sub> (30%) |} === Procedure === ---- * Upon completed transfer of the proteins to the PVDF..." current Tag: Visual edit
  • 14:2614:26, 27 February 2025 diff hist +1,505 N Western Blot: semi-dry blot Created page with "{| 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, 10 ml MeOH, fill up to 100 ml with ddH2O * Cathode Buffer preparation: 20 ml 5x Cathode buffer, 10 ml MeOH, fill up to 100 ml with ddH2O * The PVDF membrane is labeled with a pe..." Tag: Visual edit
  • 14:2414:24, 27 February 2025 diff hist 0 Western Blot: wet blot No edit summary current Tag: Visual edit
  • 14:2314:23, 27 February 2025 diff hist +1,823 N Western Blot: wet blot Created page with "{| class="wikitable" |+ !Materials ! |- |Western Transfer Buffer |dissolve 6.0 g Tris Base; 28.8 g glycine in 1 L dH<sub>2</sub>O add 430 mL methanol and degas for 30 min while stirring after degasing, add 10 mL 20% SDS and fill up to 2 L with H<sub>2</sub>O |- |Blocking Solution |TBS-T; 2% BSA; 0.05% NaN<sub>3</sub> |- |TBS-Tween |200 mL 10x TBS; 10 mL Tween; ad 2 L H<sub>2</sub>O |- | |First and second Antobody |- | |ECL-Medium |- | |H<sub>2</sub>O<sub>2</sub> (30%) |-..." Tag: Visual edit
  • 14:2114:21, 27 February 2025 diff hist +41 N File:Schema Sandwich WB.jpg No edit summary current
  • 14:1514:15, 27 February 2025 diff hist +489 N Coomassie Blue staining of gels Created page with "{| class="wikitable" |+ !Materials ! |- |Staining Solution |25% (v/v) isopropanol 10% (v/v) acetic acid 0.03% (w/v) Coomassie Brillant Blue R250 |- |Destaining Sol. |500 mL methanol; 500 mL dH<sub>2</sub>O 100 mL acetic acid |} === Procedure === ----The gel is heated in Coomassie staining solution briefly in the microwave and incubated for 30 min on a shaker. After this time it is incubated in destaining solution, so that the blue protein bands are visible against a cle..." current Tag: Visual edit
  • 14:1314:13, 27 February 2025 diff hist +2,003 N SDS-PAGE Created page with "{| class="wikitable" |+ !Materials ! |- |Separating Gel (12,5%): |3.1 mL Polyacrylamide (40%); 4.3 mL dH<sub>2</sub>O; 2.5 mL Tris (1.5 M; pH 8.8) ''Degas!'' 50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS; 10%); 15 μL TEMED |- |Stacking Gel (5%): |1.25 mL Polyacrylamide (40%); 6.15 mL dH<sub>2</sub>O; 2.5 mL Tris (0,5 M; pH 6,8) ''Degas!'' 50 μL Sodium dodecyl sulfate (SDS; 20%); 30 μL Ammonium Persulfate (APS; 10%); 15 μL TEMED |- |Marker..." current Tag: Visual edit
  • 14:1014:10, 27 February 2025 diff hist +916 N Whole cell lysates (WCLs) of eukaryotic cells Created page with "{| class="wikitable" |+ !Materials ! |- | |PBS 1X |- | |RIPA buffer |- | |Sepharose Beads |} === Procedure === ----All following steps are performed in the cold room: * Aspirate cell culture medium with a pasteur pipette * Rince attaching cells carefully with cold PBS * Add 1 mL RIPA buffer per 10 cm dish or 0.5 mL per 6 cm dish and incubate for approx. 2 min at 4°C * Carefully detach the cells with a cell scraper and transfer the lysate in a fresh tube * Mechanical s..." current Tag: Visual edit
  • 14:0814:08, 27 February 2025 diff hist +24 Methods and Protocols No edit summary Tag: Visual edit
  • 13:5313:53, 27 February 2025 diff hist +33 Measurement of endocytosis via reversible surface biotinylation No edit summary current Tag: Visual edit
  • 13:5313:53, 27 February 2025 diff hist +992 N Measurement of endocytosis via reversible surface biotinylation Created page with "{| class="wikitable" |+ !Materials ! |- |Biotinylation buffer: |PBS++ (pH 7.4) + 400 µg/ml Sulfo-NHS-SS- Biotin (freshly added) |- |Quenching buffer: |PBS++ (pH 7.4) + 100 mM Glycin |- |Stripping buffer: |50 mM Tris (pH 8.6), 100 mM NaCl, freshly added: 0.2 % BSA, 100 mM MESNa |- |Serum starvation medium: |DMEM + 0.5 % calf serum (CS) |} === Procedure === ---- * 2 x 105 cells in 6 cm dishes, serum starvation over night * Wash 3x with cold PBS++ at 4°C * Incubatio..." Tag: Visual edit
  • 13:5013:50, 27 February 2025 diff hist +1,834 N Preparation of deterg.-resist. membrane microdomains (Sucrose Gradient) Created page with "{| class="wikitable" |+ |'''Materials''' | colspan="3" | |- |Homogenisation buffer |50 mM Tris pH 7.4 |[0.5M] |▶ 10 ml |- | |2 mM MgCl<sub>2</sub> 8 % sucrose |[1M] |▶ 200 µl ▶8 g |- | |ddH<sub>2</sub>O | |▶ 89.8 ml |- |TNE buffer | colspan="2" |20 mM TrisHCL pH8 [1M] |▶ 2 ml |- | | colspan="2" |130 mM NaCl [5M] |▶ 2.6 ml |- | | colspan="2" |5 mM EDTA [0.5M] |▶ 1 ml |- | | colspan="2" |10 µg/ml Aprotinin [5mg/ml] |▶ 200 µl |- | | colspan="2" |10 µg/..." current Tag: Visual edit
  • 13:4513:45, 27 February 2025 diff hist +1,060 N Measurement of lipid raft localization via flow cytometry Created page with "{| class="wikitable" |+ !Materials ! |- | |PBS (pH 7.4) Trypsin/EDTA Triton-X100 Serum starvation medium: 0.5% calf serum (CS) FACS buffer: 46.5 ml PBS + 2.5 ml FCS (h.i) + 1 ml 5% sodium azide |} === Procedure === ---- * Add cells in poly-L-lysin coated dishes * Optionally: Serum starvation of cells over night, 37 °C * Positive control: fluorescently labelled choleratoxin for staining of Gm1 * Negative controls: staining of transferrin receptor, untransfected cells..." current Tag: Visual edit
  • 13:4113:41, 27 February 2025 diff hist +1,217 N SEAP Reporter Assay Created page with "{| class="wikitable" |+ !Materials ! |- | |Eppi centrifuge at RT Heat block @65°C Substrate buffer @+4°C pNPP powder Calf Intestine Alkaline Phosphatase (CIAP) [stock: 1μL/mL, 1:1000 dilution) Multichannel pipette and plastic box for solution Spectrophotometer (VarioSkant) |- |Assay buffer (100ml) |100 mM Glycin pH 10.4 [10 M] ▶ 1 mL 1 mM ZnCl2 [1 M] ▶ 400 μL 1 mM MgCl2 [1 M] ▶ 400 μL Adjust pH with NaOH to pH 10.4 |} === Procedure === ---- * Transfer  ..." current Tag: Visual edit
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