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>
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<p>For 250 mL M9 medium you need 175 mL sterile water (for plates add 4 g Agar-Agar as well). Then add (in the following order:</p>
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<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>
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<p> - 250 µL 100 mM CaCl2 </p>
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<p> - 300 µL 1 M CaCl2 </p>
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<p> - 2.5 mL trace salts </p>
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<p> - 100 mL 5x M9 salt solution</p>  
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<p> - store this stock solution in the dark </p>
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<p> - 250 µL MgSO4 </p>
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<p> - 20 mL 20 % glucose</p>
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<p> - 250 µL 50 mM FeCl3 / 100 mM citrate (one solution, citrate is iron carrier) </p>  
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<p> - 1 M MgSO4 </p>
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<p> - store this stock solution cold and in the dark </p>  
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<p> - 1 mL biotin (1 mg/mL)</p>
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<p> - carbon source stock solution (e.g. glucose)</p>
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<p> - 1 mL thiamin (1 mg/mL)</p>
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<p> - 50 mL 5x M9 salts stock solution </p>
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<p> - 10 mL 100x trace elements solution <p>
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<p> - 64 g L-1 Na2HPO4 * 7 H2O </p>
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<p> - 15 g L-1 KH2PO4</p>
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<p> - 2.5 g L-1 NaCl </p>
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<p> - 5 g L-1 NH4Cl </p>
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<p> - antibiotic stock solution </p>
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<p> - fill up to 250 mL with sterile water</p>
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Revision as of 11:09, 5 September 2013






Cultivation

[Media]

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

- 100 mL 5x M9 salt solution

- 20 mL 20 % glucose

- 1 M MgSO4

- 1 mL biotin (1 mg/mL)

- 1 mL thiamin (1 mg/mL)

- 10 mL 100x trace elements solution














[Buffers, Solutions]

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)



























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