Team:Tuebingen/Notebook/Protocols/mutagenesis PCR

From 2014.igem.org

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     </ul></li>
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   <li>Add 1 μl DpnI, incubation for 2 h at 37 °C</li>
   <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>

Revision as of 22:33, 14 October 2014


Mutagenesis PCR

Reagents

200 ng Template
1 µL Primer forward (2 µM)
1 µL Primer forward (2 µM)
1 µL dNTP mix (10 mM)
5 µL 10x PCR buffer
1 ng Pfu Polymerase (1 U/µL)
to 50 µL H20

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"