| 0.5 M Na2EDTA, pH 8 | Storage at RT |
| 9.3 g | Na2EDTA * 2 H2O in 40 ml A. dest. |
| 1.00 g | NaOH pellets |
| adjust to pH 8 with 5 M NaOH | |
| adjust vol. to 50 ml |
| 1.0 M MgSO4 | Storage at RT |
| 24.65 g | MgSO4 * 7 H2O |
| adjust vol. to 100 ml and autoclave |
| 5.0 M KOAc | Storage at RT |
| 49.07 g | K-acetate |
| adjust vol. to 100 ml |
| 3.0 M NaOAc (pH 5.2) | Storage at RT |
| 40.8 g | Na-acetate * 3 H2O |
| 40 ml | A. dest. |
| adjust pH to 5.2 with glacial acetic acid | |
| adjust vol. to 50 ml |
| 5.0 M NaOH (C) | Storage at RT |
| 10.0 g | NaOH |
| adjust vol. to 50 ml |
| 1.0 M NaOH (C) | Storage at RT |
| 8.0 g | NaOH |
| adjust vol. to 200 ml |
| 3.5 M HCl (C) | Storage at RT |
| 95 ml | HCl (25 % - ρ = 1.08; 7.4 M) |
| adjust vol. to 200 ml |
| 1.0 M HCl (C) | Storage at RT |
| 30 ml | HCl (25 %) |
| adjust vol. to 210 ml |
| 1.5 M NaCl | Storage at RT |
| 8.75 g | NaCl |
| adjust vol. to 100 ml |
| 10 % SDS | Storage at RT, dark |
| 1.00 g | SDS (use mask during weighing) |
| adjust vol. to 10 ml |
| 1.0 M Tris-HCl, pH 7.5 | Storage at RT |
| 12.1 g | Tris-base |
| 80.0 ml | A. dest. |
| adjust to pH 7.5 with 5 M HCl | |
| adjust vol. to 100 ml and autoclave |
| 1.0 M Tris-HCl, pH 8.0 | Storage at RT |
| 12.1 g | Tris-base |
| 80.0 ml | A. dest. |
| adjust to pH 8.0 with 5 M HCl | |
| adjust vol. to 100 ml and autoclave |
| 10x TE | Storage at RT |
| 5.0 ml | 1 M Tris, pH 8 (= 100 mM) |
| 1.0 ml | 0.5 M EDTA, pH 8 (= 10 mM) |
| adjust vol. to 50 ml |
| 20x TAE | Storage at RT |
| 96.9 g | Tris base (= 800 mM) |
| 33.0 g | Na-acetate (= 800 mM) |
| 14.9 g | Na2EDTA * 2 H2O (= 40 mM) |
| 800 ml | A. dest. |
| adjust to pH 8.3 with glacial acetic acid (C) | |
| adjust vol. to 1,000 ml |
| 5x TBE | Storage at RT |
| 54 g | Tris base (= 450 mM) |
| 27.5 g | Boric acid (= 450 mM) (T) |
| 3.75 g | Na2EDTA * 2 H2O (= 10 mM) |
| adjust vol. to 1,000 ml |
| 20x SB | Storage at RT |
| 8 g | NaOH (= 200 mM) (C) |
| 45 g | Boric acid (= 730 mM) (T) |
| adjust vol. to 1,000 ml (pH 8.0 - 8.3) |
| 1 M Acetic acid (C) | Storage at RT |
| 12 ml | Glacial acetic acid |
| adjust vol. to 200 ml |
| Sol A | Storage at 4 °C |
| 0.46 g | glucose (Glc) |
| 1.25 ml | 1 M Tris, pH 8 |
| 1.00 ml | 0.5 M EDTA, pH 8 |
| adjust vol. to 50 ml and autoclave |
| Sol B | Storage at RT |
| 1.6 ml | 5 M NaOH (or 0.4 g NaOH pellets) |
| 34.4 ml | A. dest. |
| 4.00 ml | 10 % SDS |
| Sol C | Storage at 4 °C |
| 60.0 ml | 5 M KOAc |
| 11.5 ml | glacial acetic acid (C) |
| 28.5 ml | A. dest. |
| Gro conservation soln | Storage at 4 °C |
| 52.0 g | glycerol (Gro) |
| 2.0 ml | 1 M Tris, pH 8 |
| 8.0 ml | 1 M MgSO4 |
| adjust vol. to 80 ml and autoclave |
| GYT | Storage at 4 °C |
| 5.00 g | glycerol (Gro) |
| 0.06 g | yeast extract |
| 0.13 g | tryptone |
| adjust vol. to 50 ml and autoclave |
| 10 % Gro | Storage at 4 °C |
| 40.0 g | glycerol (Gro) |
| adjust vol. to 400 ml and autoclave |
| LB-medium | Storage at RT |
| 500 ml | A. dest. |
| 10 g | tryptone |
| 5 g | yeast extract |
| 10 g | NaCl (= LB-Miller) LB-Lennox: 5 g/l NaCl (for salt sentive antibiotics like Zeocin) |
| 200 µl | 5 M NaOH (pH 7.0) if necessary, adjust pH before adding agar |
| 15 g | Agar (only for agar plates) |
| adjust vol. to 1,000 ml and autoclave | |
| pour agar plates after cooling down to ~ 50 ºC to avoid condensed water add antibiotics etc. only after autoclaving when cooled down LB = Lysogeny broth The original recipe contains 1 g/l Glc |
| Amp stock (Xn) | Storage at -20 °C |
| 25.0 mg/ml | ampicillin-Na in A. dest. |
| sterilize by filtration | |
| add 400 µl to 100 ml LB => 100 µg/ml | |
| store LB-Amp and LB-Amp/X-Gal plates @ 4 °C |
| Kan stock | Storage at -20 °C |
| 50.0 mg/ml | kanamycin in A. dest. |
| sterilize by filtration | |
| add 100 µl to 100 ml LB => 50 µg/ml |
| Cm stock (T, F) | Storage at -20 °C |
| 100.0 mg/ml | chloramphenicol in EtOH |
| add 25 µl to 100 ml LB => 25 µg/ml |
| Zeo stock | Storage at -20 °C |
| 100.0 mg/ml | Zeocin in A. dest. (ready) |
| add 25 µl to 100 ml LB => 25 µg/ml Beware: LB-Zeocin requires low salt LB (5 g/l) |
| AmB stock | Storage at -20 °C |
| 25.0 mg/ml | amphotericin B in sterile DMSO |
| add 100 µl to 100 ml LB => 2.5 µg/ml | |
| store LB-AmB @ 4 °C |
| IPTG stock | Storage at -20 °C |
| 0.24 g | isopropylthio-ß-D-galactoside |
| 10.0 ml | A. dest. |
| sterilise by filtration | |
| add 100 µl to 100 ml LB => 0.1 mM |
| X-Gal stock (Xn, T) | Storage at -20 °C |
| 40.0 mg/ml | 5-bromo-4-chloro-3-indoyl-ß-D-galactoside in Dimethylformamide, DMF (T) |
| add 100 µl to 100 ml LB => 40 µg/ml |
When preparing more than 20 agarplates of one type of medium, it is annoying to write on each agarplate the type of medium. As it is easier and much faster to make lines at the side of a tower of agarplates, the bar code for the CBL is the following:
| LB medium | |
| | | LB-Amp |
| || | LB-Amp/XGal |
| ||| | LB-Kan |
| |||| | LB-Cm |
| ||||| | LB-Zeo |
| Proteinase K | Storage at -20 °C |
| 2 mg | proteinase K |
| 200 µl | A. dest. |
| => 10 mg/ml |
| RNase A | Storage at -20 °C |
| 5 mg | RNase A |
| 500 µl | 1x TE |
| => 10 mg/ml |
| BSA | Storage at -20 °C |
| 20 mg | bovine serum albumin |
| 1 ml | A. dest. |
| => 20 mg/ml |
| Glycogen | Storage at -20 °C |
| 20 mg/ml | ready |
| 100 mM DTT (Xn) | Storage at -20 °C |
| 154 mg | 1,4-dithio-DL-threitol, DTT |
| 10 ml | sterile A. dest |
| aliquot in 500 µl portions use final concentrations of 1 mM |
| λPst-size marker | Storage at -20 °C |
| 167 µl | λ-DNA, dam-, dcm- (300 µg/ml) |
| 10 µl | PstI (10 U/µl) |
| 40 µl | O+-buffer (10x) |
| 200 µl | A. dest. |
| incubate 2 h @ 37 °C heat inactivate 15 min @ 65 °C |
|
| 100 µl | 6x Loading dye |
| final DNA concentration: 0,1 µg/µl with 5 µl the 1 kbp-band represents ~ 10 ng DNA |
| 100 mM PMSF (T) | Storage at -20 °C (dark) |
| 17.4 mg | Phenylmethylsulfonyl fluoride, PMSF |
| 1 ml | DMSO |
| aliquot in 100 µl portions use final concentrations of 0.1-1 mM (1-10 µl in 1 ml cell extract). Add only immeadiately prior use due to its short half-life at neutral-basic pH (@ pH 7.5 ~30 min) PMSF is a Serine protease inhibitor. DTT or β-mercaptoethanol contra-act PMSF by reducing thiol groups less toxic alternative may be: AEBSF-HCl |
| Cell Lysis Buffer (preliminary) | Storage at 4 °C |
| 44 ml | PBS [when too much salt: 20 mM Tris-HCl, pH 7.5: 1 ml 1 M Tris-HCl, pH 7.5 + 43 ml A. dest.] |
| 5 ml | glycerol |
| 0.5 ml | Triton X-100 |
| 0.5 ml | 0.5 M EDTA |
| add 1-10 µl PMSF (T) to 1 ml Cell lysis buffer immediately prior use. Wear gloves during preparation and use to avoid contamination with skin protein. |
| 0.5% Bromophenol blue | Storage at 4 °C |
| 50 mg | bromophenol blue |
| 100 µl (5 ml?) | ethanol (96%) |
| dissolve and adjust vol. to 10 ml with A. dest. |
| 5X SDS Sample Buffer | (SDS Reducing Buffer, Loading Buffer) Storage at RT, dark |
| 3.5 ml | 0.5 M Tris-HCl, pH 6.8 (= Stacking Gel Buffer) |
| 4 ml | glycerol |
| 2.0 ml | 10 % SDS |
| 0.25 ml | 0.5 % bromophenol blue |
| add 50 µl mercaptoethanol (T) to 950 µl Sample buffer prior use. Add to 4 vol. sample 1 vol. Sample buffer and heat up immediately to 95 °C for 3-5 min. Wear gloves during preparation and use to avoid contamination with skin protein. |
| 5X Protein Loading Buffer | (Ready to use, Fermentas R0891) Storage at -20 °C |
| 0.313 M | Tris-HCl, pH 6.8 |
| 50% | glycerol |
| 10% | SDS |
| 0.05% | bromophenol blue |
| Thawn up components to RT, vortex to dissolve al precipitates First use: Aliquot Loading Buffer into 1 ml Add 1/5 vol. Loading Buffer to sample (50 µl to 200 µl sample) Add 1/20 vol. Reducing Agent (2 M DTT; Xn) to sample (10 µl to 200 µl sample) Heat up sample (standard: 3-5 min to 95 °C) |
| Resolving Gel Buffer (1.5 M Tris, pH 8.8) | Storage at 4 °C |
| 81.69 g | Tris base |
| 300 ml | A. dest. |
| adjust pH to 8.8 with 5 N HCl adjust vol. to 450 ml (enough for ~90 mini-gels) |
| Stacking Gel Buffer (0.5 M Tris, pH 6.8) | Storage at 4 °C |
| 6.0 g | Tris base |
| 60 ml | A. dest. |
| adjust pH to 6.8 with 5 N HCl adjust vol. to 100 ml (enough for > 150 Mini-gels) |
| 40% Acrylamide/Bisacrylamide, 37.5:1 (T) | Storage at 4 °C |
| 389.6 g/l | acrylamide |
| 10.4 g/l | N,N'-methylene-bis-acrylamide |
| Due to the high toxicity of acrylamide (specially the powder), it it useful to buy Ready to use solution, e.g.: - BioRad 161-0148 |
| 10% APS (O, Xn) | daily fresh |
| 10 mg | ammonium persulfate, APS |
| 100 µl | A. dest. |
| enough for preparing 1 mini-gel |
| Prestained Size Marker | -20 °C |
| 500 µl | Kaleidoscope Prestained Standard (Biorad #161-0324) |
 |
Warm soln. to 37 °C for 1 min and dissove any solids use 5 µl for a mini-gel (visible after blotting) or 10 µl (visible during electrophoresis) Prestained standrds are not further stained by Coomassie blue. Myosin (blue, 194-198.5 kDa) β-galactosidase (magenta, 111-126.3 kDa) BSA (green, 59-87.8 kDa) Carbonic anhydrase (violet, 30-38.2 kDa) Soybean trypsin inhibitor (orange, 24.9-31.3 kDa) Lysozyme (red, 12.4-17 kDa) Aprotinin (blue, 6.4-7 kDa) |
| 10X Running Buffer | (Electrode Buffer, Reservoir Buffer) Storage at 4 °C |
| 30.3 g | Tris base |
| 144 g | glycine |
| 10 g | SDS |
| 1 l | A. dest. |
| Do not adjust pH (pH ~ 8.3 - 8.8 (?)) If precipitate, warm up to RT Dilute 70 ml 10x Running Buffer to 700 ml with A. dest. for running 1-2 gels Dilute 100 ml 10x Running Buffer to 1,000 ml with A. dest. for running 3-4 gels Mix well before use |
| Coomassie blue G250 | (Classic) Storage at 4 °C, dark |
| 0.1 g | Coomassie blue G250 |
| 1 ml | methanol (or ethanol) |
| Add 1 ml [250 µl] to 50 ml Staining Solution (0.25% [0.05%]) G250 is less toxic than R250 |
| Colloidal Coomassie Brilliant Blue G-250 | (CBB-G250 Staining Solution) Storage at 4 °C, dark |
| 500 ml | A. dest. |
| 50 g | ammonium sulfate, (NH4)2SO4 dissolve |
| 20 ml | phosphoric acid add alternative: 10 ml 3.5 M HCl? |
| 100 ml | ethanol add |
| 0.2 g | Coomassie Brillant Blue G250 add |
| Prepare in this orden! Final solution is colloidal greenish-dark Do not filtrate Reuseable until getting blue |
| 1X Universal Transfer-buffer | Storage at 4 °C |
| 3.03 g | Tris base (25 mM) |
| 14.4 g | glycine (192 mM) |
| 100-200 ml | MeOH (= 10-20%): use 10% for transfer of big proteins MeOH can be omitted when using PVDF-membranes |
| 5-10 ml | 10% SDS (0.05-0.1%) |
| ad. 1 l | A. dest. |
| Do not adjust pH (pH ~ 8.3) 950 ml are required for one run with the Mini-Trans-Blot system (Biorad) Mix well before use |
| 10X TBS | Storage at 4 °C |
| 12.1 g | Tris base |
| 80.0 g | NaCl |
| 800 ml | A. dest. |
| ajust pH to 7.3 with 5 N HCl adjust vol. to 1,000 ml |
| 10% Tween 20 | Storage at 4 °C, max. 2 month |
| 5 ml | Tween 20 (heat up to 50 °C for easier pipetting) |
| ad 50 ml | A. dest. |
| TBS(T) | Storage at 4 °C |
| 100 ml | 10X TBS |
| (5 ml) | 10% Tween 20 (= 0.05%) as Tween is *very* viscose it would be difficult to pipette 0.5 ml of Tween directly |
| ad 1,000 ml | A. dest. |
| Blotto Blocking-buffer |
Storage at 4 °C. Prepare daily fresh |
| 10 ml | 10X TBS |
| 5 g | non-fat milk-powder (= 5%) |
| (1 ml) | 10% Tween 20 (= 0.05%) as Tween is *very* viscose it would be difficult to pipette 0.25 ml of Tween directly |
| ad 100 ml | A. dest. |
| enough for 2 membranes (?) add 25 µl Cm stock (T, F; 100 mg/ml; -20 °C) to inhibit bacterial growth? |
