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Team:Bielefeld-Germany/Labjournal/Cultivation
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Revision as of 14:42, 5 September 2013
Cultivation
Chloramphenicol stock solution
-Solubilize 20 mg mL-1 Chloramphenicol in 100 % Ethanol
-Store at -20 °C
DNA loading buffer
- 50 % (v/v) glycerol
- 1 mM EDTA
- 0.1 % (w/v) bromphenol blue
- Solve in ddH2O
LB medium
- 10 g Trypton
- 5 g yeast extract
- 10 g NaCl
- 12 g Agar-Agar (for plates)
- Adjust pH to 7.4
M9 medium
For 1 L M9 mineral medium you need 867 mL sterile water (for plates add 16 g Agar-Agar as well). Then add (in the following order:
- 300 µL 1 M CaCl2
| 1 M CaCl2 | 1 M CaCl2-H20 | 14.70 g/100 mL |
- 100 mL 5x M9 salt solution
| 10x M9 salt solution | |
|---|---|
| Na2HPO4-2H2O | 75.2 g/L |
| KH2PO4 | 30 g/L |
| NaCl | 5 g/L |
| NH4Cl | 5 g/L |
- 20 % glucose
| 20 % Glucose | 20 % (w/v)glucose | 200 g/L |
For 500 mL stock solution add 100 g glucose to 440 mL water. Autoclave for 15 at 121° C
- 1 M MgSO4
| MgSO4 | 1 M MgSO4-7H20 | 24.65 g/100 mL |
- 1 mL biotin (1 mg/mL)
| Biotin (1 mg/mL) | biotin (1 mg/mL) | 50 mg/50 mL |
For 50 mL stock solution dissolve 50 mg biotin in 45 mL water. Add water to a final volume of 50 mL. Sterilize the solution over a 0.22-µm filter. Prepare 1 mL aliquots and store at -20°.
- 1 mL thiamin (1 mg/mL)
| Thiamin (1 mg/mL) | thiamin-HCl (1 mg/mL) | 50 mg/50 mL |
For 50 mL stock solution dissolve 50 mg thiamin-HCl in 45 mL water. Add water to a final volume of 50 mL. Sterilize the solution over a 0.22-µm filter. Prepare 1 mL aliquots and store at -20°.
- 10 mL 100x trace elements solution
| 100x trace elements solution | EDTA | 5 g/L | 13.4 mM |
| FeCl3-6H2O | 0.83 g/L | 3.1 mM | |
| ZnCl2 | 84 mg/L | 0.62 mM | |
| CuCl2-2H20 | 13 mg/L | 76 µM | |
| CoCl2-H2O | 10 mg/L | 42 µM | |
| H3BO3 | 10 mg/L | 162 µM | |
| MnCl2-4H20 | 1.6 mg/L | 8.1 µM |
Dissolve 5g EDTA in 800 mL water and adjust the pH to 7.5 with NaOH. Then add the other components in the quantities mentioned below and add water to a final volume of 1L. Sterilize the solution over a 0.22µm filter
| 100x trace elements solution | EDTA | 5 g/L | 13.4 mM |
| 498 mg | FeCl3(anhydrous) | ||
| 84 mg | ZnCl2 | ||
| 765 µL | 0.1 M CuCl2-2H20 | 1.70 g/100 mL | |
| 210 µL | 0.2 M CoCl2-H2O | 4.76 g/100 mL | |
| 1.6 mL | 0.1 M H3BO3 | 0.62 g/100 mL | |
| 8.1 µL | 1M MnCl2-4H20 | 19.8 g/100 mL |
TAE buffer
For 1 L of 50 x TAE buffer you need:
- 242.48 g Tris
- 41.02 g Sodiumacetate
- 18.612 g EDTA
- Adjust pH to 7.8 with acetic acid
- Solve in dH2O
10 mL of the stock is diluted in 1 L dH2O for the gel electrophoresis (0.5 x TAE buffer)
