Team:Tuebingen/Notebook/Protocols/restriction

From 2014.igem.org

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    <td style="text-align: center">0.5 µL</td>
 
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    <td>Pfu Polymerase (1 U/µL)</td>
 
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    <td style="text-align: center">to 50 µL</td>
 
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    <td>H20</td>
 
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<h4>Procedure</h4>
<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>
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Revision as of 23:03, 14 October 2014


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"