Team:Bielefeld-Germany/Labjournal/Cultivation

From 2013.igem.org

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<div id=globalwrapper style="padding-left:20px; padding-right:20px;">
<br><br>
<br><br>
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<h1 style="color:#ff6600;">Cultivation</h1>
 +
<br>
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<div class="sds_headline">
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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="sds" >
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<h3>Sodium dodecyl sulfate polyacrylamide gel electrophoresis</h3>
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<br><b><u>Pouring the polyacrylamide gel</u></b>
 +
<p>- Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED</p>
 +
<p>- Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel</p>
 +
<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>
 +
<p>- Leave the separating gel at room temperature for >60 minutes to polymerize</p>
 +
<p>- Remove isopropanol and wait until the surface is dry</p>
 +
<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>
 +
<p>- Leave the gel at room temperature for >60 minutes to polymerize</p>
 +
<br>
 +
<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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</style>
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<br><b><u>Preparing the sample</u></b>
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<p>- Mix your protein mixture 4:1 with Laemmli-buffer (30 mL protein solution + 10 mL Laemmli-buffer)</p>
 +
<p>- Heat for 5 minutes at 95 °C</p>
 +
<br>
 +
<br><b><u>Running the gel</u></b>
 +
<p>- Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber</p>
 +
<p>- Remove comb without destroying the gel pocket</p>
 +
<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>
 +
<p>- Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel</p>
 +
<p>- Raise amperage up to 20 mA for running the separating gel</p>
 +
<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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<h1>Cultivation</h1>
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<br><br>
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<div class="media_1_headline">
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<a name="media_1"><span style="color:#ff6600">[Media]</span>Chloramphenicol stock solution </span></a>
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</div>
</div>
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<div class="media_1">
 
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Solubilize 20 mg mL-1 Chloramphenicol in 100 % Ethanol
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-
Store at -20 °C
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<!--
<!--
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<p> • A</p>
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<tr> <th>Reagent</th>         <th>1x</th> <th>8x</th> </tr>
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<p> • I</p>
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<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
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<p> • A</p>
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<tr> <td>dNTPs (10mM)</td>  <td align="right">2.0</td> <td align="right">17.6</td> </tr>
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<p> • I</p>
-
<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
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<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
+
-
<tr> <td>Template (16.5ng)</td> <td align="right">3.0</td> <td align="right">26.4</td> </tr>
+
-
<tr> <td>DMSO</td>         <td align="right">4.0</td>    <td align="right">35.2</td> </tr>
+
-
<tr> <td>Phusion (0.5u)</td>        <td align="right">1.0</td> <td align="right">8.8</td> </tr>
+
-
<tr> <td>H2O</td>          <td align="right">26.0</td> <td align="right">228.8</td> </tr>
+
-
</table>
 
-
 
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<table border=1 style="float:left; margin-left:20px">
 
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<tr><th colspan="5">Program Phusion</th></tr>
 
-
<tr align="center"><td>98 °C</td><td>98 °C</td><td>68 °C</td><td>72 °C</td><td>72 °C</td></tr>
 
-
<tr align="center"><td>30s</td> <td>10s</td><td>30s</td> <td>1:35</td><td>6:00</td></tr>
 
-
<tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr>
 
-
<tr><td colspan="5">
 
-
<b><u>Notes:</u></b>
 
-
<br>Size: 1800bp
 
-
<br>Primer1:
 
-
<br>Primer2:
 
-
<br>
 
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</td></tr>
 
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</table>
 
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<br><br><br><br><br><br><br><br><br><br><br><br><br>
 
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<br><br><br>
</body>
</body>
</html>
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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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