Team:Bielefeld-Germany/Labjournal/Cultivation

From 2013.igem.org

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<h1>Cultivation</h1>
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<a name="media"><span style="color:#ff6600">[Media]</span></a>
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<h1 style="color:#ff6600;">Cultivation</h1>
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<br>
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<a name="sds"><span style="color:#ff6600; padding-left=22px">[SDS-PAGE]</span></a>
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</div>
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<div class="media">
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<div class="sds" >
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<br><b><u> Chloramphenicol stock solution</u></b>
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-
<p>-Solubilize 20 mg mL-1 Chloramphenicol in 100 % Ethanol </p>
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-
<p>-Store at -20 °C</p>
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<br><b><u> DNA loading buffer</u></b>
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<h3>Sodium dodecyl sulfate polyacrylamide gel electrophoresis</h3>
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<p> - 50 % (v/v) glycerol </p>
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<br><b><u>Pouring the polyacrylamide gel</u></b>
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<p> - 1 mM EDTA </p>
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<p>- Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED</p>
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<p> - 0.1 % (w/v) bromphenol blue </p>
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<p>- Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel</p>
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<p> - Solve in ddH2O</p>
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<p>- Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel</p>
 +
<p>- Layer isopropanol on top of the ge</p>
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<p>- Leave the separating gel at room temperature for >60 minutes to polymerize</p>
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<p>- Remove isopropanol and wait until the surface is dry</p>
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<p>- Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form</p>
 +
<p>- Insert comb without getting bubbles stuck underneath</p>
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<p>- Leave the gel at room temperature for >60 minutes to polymerize</p>
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<br>
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<p><i>For storage:</i></p>
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<p style="padding-left:44px;"> -Remove sealing and store the gel wrapped in moistened paper towel at 4°C</p>
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<br>
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<br><b><u> LB medium</u></b>
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<br><b><u>Preparing the sample</u></b>
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<p> - 10 g Trypton </p>
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<p>- Mix your protein mixture 4:1 with Laemmli-buffer (30 mL protein solution + 10 mL Laemmli-buffer)</p>
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<p> - 5 g yeast extract </p>
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<p>- Heat for 5 minutes at 95 °C</p>
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<p> - 10 g NaCl </p>
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<br>
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<p><i> - 12 g Agar-Agar (for plates) </p></i>
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<p> - Adjust pH to 7.4</p>
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<br><b><u>M9 medium</u></b>
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<br><b><u>Running the gel</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>- Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber</p>
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<p> - 250 µL 100 mM CaCl2 </p>
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<p>- Remove comb without destroying the gel pocket</p>
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<p> - 2.5 mL trace salts </p>
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<p>- Pipet the sample into the gel pockets, adjusting the volume according to the amount of protein in your sample. Make sure to include a lane with molecular    weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of your sample</p>
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<p> - store this stock solution in the dark </p>
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<p>- Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel</p>
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<p> - 250 µL MgSO4 </p>
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<p>- Raise amperage up to 20 mA for running the separating gel</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>- When the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm stop the electrophoresis by turning off the power supply</p>
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<p> - store this stock solution cold and in the dark </p>
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<p> - carbon source stock solution (e.g. glucose)</p>  
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<p> - 50 mL 5x M9 salts stock 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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<br><b><u>TAE buffer</u></b>
 
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<p>For 1 L of 50 x TAE buffer you need:</p>
 
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<p> - 242.48 g Tris </p>
 
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<p> - 41.02 g Sodiumacetate</p>
 
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<p> - 18.612 g EDTA </p>
 
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<p> - Adjust pH to 7.8 with acetic acid </p>
 
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<p> - Solve in dH2O </p>
 
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<p><i>10 mL of the stock is diluted in 1 L dH2O for the gel electrophoresis (0.5 x TAE buffer) </i></p>
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<p> • I</p>
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Latest revision as of 11:34, 26 September 2013







Cultivation


[SDS-PAGE]

Sodium dodecyl sulfate polyacrylamide gel electrophoresis


Pouring the polyacrylamide gel

- Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED

- Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel

- Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel

- Layer isopropanol on top of the ge

- Leave the separating gel at room temperature for >60 minutes to polymerize

- Remove isopropanol and wait until the surface is dry

- Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form

- Insert comb without getting bubbles stuck underneath

- Leave the gel at room temperature for >60 minutes to polymerize


For storage:

-Remove sealing and store the gel wrapped in moistened paper towel at 4°C



Preparing the sample

- Mix your protein mixture 4:1 with Laemmli-buffer (30 mL protein solution + 10 mL Laemmli-buffer)

- Heat for 5 minutes at 95 °C



Running the gel

- Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber

- Remove comb without destroying the gel pocket

- Pipet the sample into the gel pockets, adjusting the volume according to the amount of protein in your sample. Make sure to include a lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of your sample

- Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel

- Raise amperage up to 20 mA for running the separating gel

- When the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm stop the electrophoresis by turning off the power supply






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