Team:Bielefeld-Germany/Labjournal/Cultivation
From 2013.igem.org
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<br><b><u>M9 medium</u></b> | <br><b><u>M9 medium</u></b> | ||
<p>For 1 L <b>M9 mineral medium</b> you need 867 mL sterile water <i>(for plates add 16 g Agar-Agar as well)</i>. Then add (in the following order:</p> | <p>For 1 L <b>M9 mineral medium</b> you need 867 mL sterile water <i>(for plates add 16 g Agar-Agar as well)</i>. Then add (in the following order:</p> | ||
- | <p> - 300 µL 1 M CaCl2 | + | <p> - 300 µL 1 M CaCl2 </p> |
- | + | <table border=1; style="margin-left:22px"> | |
- | </ | + | <col width="150"> |
- | + | <col width="150"> | |
+ | <col width="100"> | ||
+ | <tr> <td align="center"><b>1 M CaCl2</b></td> <td align="center">1 M CaCl2-H20</td> <td align="center">14.70 g/100 mL</td> </tr> | ||
+ | </table> | ||
<p> - 100 mL 5x M9 salt solution</p> | <p> - 100 mL 5x M9 salt solution</p> | ||
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</table> | </table> | ||
<br> | <br> | ||
- | <p> - 20 % glucose | + | <p> - 20 % glucose</p> |
<table border=1; style="margin-left:22px"> | <table border=1; style="margin-left:22px"> | ||
<col width="150"> | <col width="150"> | ||
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<p> For 500 mL stock solution add 100 g glucose to 440 mL water. Autoclave for 15 at 121° C</p> | <p> For 500 mL stock solution add 100 g glucose to 440 mL water. Autoclave for 15 at 121° C</p> | ||
<p> - 1 M MgSO4 </p> | <p> - 1 M MgSO4 </p> | ||
- | + | <table border=1; style="margin-left:22px"> | |
+ | <col width="150"> | ||
+ | <col width="150"> | ||
+ | <col width="100"> | ||
+ | <tr> <td align="center"><b>MgSO4</b></td> <td align="center">1 M MgSO4-7H20</td> <td align="center">24.65 g/100 mL</td> </tr> | ||
+ | </table> | ||
<p> - 1 mL biotin (1 mg/mL)</p> | <p> - 1 mL biotin (1 mg/mL)</p> | ||
+ | <table border=1; style="margin-left:22px"> | ||
+ | <col width="150"> | ||
+ | <col width="150"> | ||
+ | <col width="100"> | ||
+ | <tr> <td align="center"><b>Biotin (1 mg/mL)</b></td> <td align="center">biotin (1 mg/mL)</td> <td align="center">50 mg/50 mL</td> </tr> | ||
+ | </table> | ||
+ | <p> 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°.</p> | ||
<p> - 1 mL thiamin (1 mg/mL)</p> | <p> - 1 mL thiamin (1 mg/mL)</p> | ||
+ | <table border=1; style="margin-left:22px"> | ||
+ | <col width="150"> | ||
+ | <col width="150"> | ||
+ | <col width="100"> | ||
+ | <tr> <td align="center"><b>Thiamin (1 mg/mL)</b></td> <td align="center">thiamin-HCl (1 mg/mL)</td> <td align="center">50 mg/50 mL</td> </tr> | ||
+ | </table> | ||
+ | <p> 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°.</p> | ||
<p> - 10 mL 100x trace elements solution <p> | <p> - 10 mL 100x trace elements solution <p> | ||
- | + | <table border=0; style="margin-left:22px"> | |
+ | <col width="150"> | ||
+ | <col width="100"> | ||
+ | <col width="100"> | ||
+ | <col width="100"> | ||
+ | <tr> <td align="center"><b>100x trace elements solution </b></td> <td align="center">EDTA</td> <td align="center">5 g/L</td> <td align="center">13.4 mM</td></tr> | ||
+ | <tr> <td align="center"></td> <td align="center">FeCl3-6H2O</td> <td align="center">0.83 g/L</td> <td align="center">3.1 mM</td></tr> | ||
+ | <tr> <td align="center"></td> <td align="center">ZnCl2</td> <td align="center">84 mg/L</td> <td align="center">0.62 mM</td></tr> | ||
+ | <tr> <td align="center"></td> <td align="center">CuCl2-2H20</td> <td align="center">13 mg/L</td> <td align="center">76 µM</td></tr> | ||
+ | <tr> <td align="center"></td> <td align="center">CoCl2-H2O</td> <td align="center">10 mg/L</td> <td align="center">42 µM</td></tr> | ||
+ | <tr> <td align="center"></td> <td align="center">H3BO3</td> <td align="center">10 mg/L</td> <td align="center">162 µM</td></tr> | ||
+ | <tr> <td align="center"></td> <td align="center">MnCl2-4H20</td> <td align="center">1.6 mg/L</td> <td align="center">8.1 µM</td></tr> | ||
+ | </table> | ||
+ | <p> 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</p> | ||
+ | <table border=0; style="margin-left:22px"> | ||
+ | <col width="100"> | ||
+ | <col width="150"> | ||
+ | <col width="100"> | ||
+ | <tr> <td align="center"><b>100x trace elements solution </b></td> <td align="center">EDTA</td> <td align="center">5 g/L</td> <td align="center">13.4 mM</td></tr> | ||
+ | <tr> <td align="center">498 mg</td> <td align="center">FeCl3(anhydrous)</td> <td align="center"></td></tr> | ||
+ | <tr> <td align="center">84 mg</td> <td align="center">ZnCl2</td> <td align="center"></td></tr> | ||
+ | <tr> <td align="center">765 µL</td> <td align="center">0.1 M CuCl2-2H20</td> <td align="center">1.70 g/100 mL</td> </tr> | ||
+ | <tr> <td align="center">210 µL</td> <td align="center">0.2 M CoCl2-H2O</td> <td align="center">4.76 g/100 mL</td></tr> | ||
+ | <tr> <td align="center">1.6 mL</td> <td align="center">0.1 M H3BO3</td> <td align="center">0.62 g/100 mL</td></tr> | ||
+ | <tr> <td align="center">8.1 µL</td> <td align="center"> 1M MnCl2-4H20</td> <td align="center">19.8 g/100 mL</td></tr> | ||
+ | </table> | ||
<br><br><br><br> | <br><br><br><br> |
Revision as of 14:41, 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)