Team:Tuebingen/Notebook/Protocols/mutagenesis PCR

From 2014.igem.org

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<div id="TueContent">
<div id="TueContent">
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<h1>Protocols</h1>
<h3>Mutagenesis PCR</h3>
<h3>Mutagenesis PCR</h3>
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   <tr>
   <tr>
     <td style="text-align: center">1 µL</td>
     <td style="text-align: center">1 µL</td>
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     <td>Primer forward (2 µM)</td>
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     <td>Primer reverse(2 µM)</td>
   </tr>
   </tr>
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   <tr>
   <tr>
     <td style="text-align: center">to 50 µL</td>
     <td style="text-align: center">to 50 µL</td>
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     <td>H20</td>
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     <td>H<sub>2</sub>O</td>
   </tr>
   </tr>
</table>
</table>
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<p>&nbsp;</p>
<h4>Procedure</h4>
<h4>Procedure</h4>
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       <li>infinite / 20 °C </li>
       <li>infinite / 20 °C </li>
     </ul></li>
     </ul></li>
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   <li>Add 1μl DpnI, incubation for 2h at 37°C</li>
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   <li>Add 1 μl DpnI, incubation for 2 h at 37 °C</li>
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   <li><a href="https://2014.igem.org/Team:Tuebingen/Notebook/Protocols/transformation">Transformation in <i>E. coli</i> </a> (5 μl)</li>
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   <li><a href="https://2014.igem.org/Team:Tuebingen/Notebook/Protocols/transformation">Transformation in <i>E. coli</i></a> (5 μl)</li>

Latest revision as of 15:33, 17 October 2014


Protocols

Mutagenesis PCR

Reagents

200 ng Template
1 µL Primer forward (2 µM)
1 µL Primer reverse(2 µM)
1 µL dNTP mix (10 mM)
5 µL 10x PCR buffer
1 ng Pfu Polymerase (1 U/µL)
to 50 µL H2O

 

Procedure

  1. Run cycler: PCR-program
    • 5 min / 95 °C
    • 1 min / 94 °C
    • 1 min / 56 °C
    • 2 min per kb / 68 °C
    • 10 min / 68 °C
    • infinite / 20 °C
  2. Add 1 μl DpnI, incubation for 2 h at 37 °C
  3. Transformation in E. coli (5 μl)

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