Team:Tuebingen/Notebook/Protocols/restriction

From 2014.igem.org

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<h1>Protocols</h1>
<h3>Preparative restriction digests</h3>
<h3>Preparative restriction digests</h3>
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<h4>Reagents</h4>
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<table border="0">
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  <colgroup>
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    <col width="100">
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    <col width="300">
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  </colgroup>
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 +
  <tr>
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    <td style="text-align: center">min. 2 µg</td>
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    <td>Plasmid DNA</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">2 µL</td>
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    <td>of each restriction enzyme</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">10 µL</td>
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    <td>10x NE buffer 2</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">1 µL</td>
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    <td>100x BSA</td>
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  </tr>
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  <tr>
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    <td style="text-align: center">to 100 µL</td>
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    <td>H<sub>2</sub>O</td>
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  </tr>
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</table>
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<p>&nbsp;</p>
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<h4>Procedure</h4>
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<ol>
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  <li>After mixing the reagents in a 1.5 ml Eppendorf-tube: Incubation at 37 °C overnight.  </li>
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  <li>Heat inactivate the restriction enzymes at 80 °C for 20 min.  </li>
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  <li>Treat the plasmid backbones with Antarctic Phosphatase (NEB-protocol).  </li>
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  <li>Separation via gel electrophoresis and gel extraction.  </li>
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</ol>
</div>
</div>

Latest revision as of 15:34, 17 October 2014


Protocols

Preparative restriction digests

Reagents

min. 2 µg Plasmid DNA
2 µL of each restriction enzyme
10 µL 10x NE buffer 2
1 µL 100x BSA
to 100 µL H2O

 

Procedure

  1. After mixing the reagents in a 1.5 ml Eppendorf-tube: Incubation at 37 °C overnight.
  2. Heat inactivate the restriction enzymes at 80 °C for 20 min.
  3. Treat the plasmid backbones with Antarctic Phosphatase (NEB-protocol).
  4. Separation via gel electrophoresis and gel extraction.

Retrieved from "http://2014.igem.org/Team:Tuebingen/Notebook/Protocols/restriction"