Team:Tuebingen/Notebook/Protocols/mutagenesis PCR

From 2014.igem.org


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)

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