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Team:Tuebingen/Notebook/Protocols/mutagenesis PCR
From 2014.igem.org
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<td style="text-align: center">1 µL</td> | <td style="text-align: center">1 µL</td> | ||
| - | <td>Primer | + | <td>Primer reverse(2 µM)</td> |
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<h4>Procedure</h4> | <h4>Procedure</h4> | ||
Revision as of 23:10, 14 October 2014
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 | H20 |
Procedure
- 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
- Add 1 μl DpnI, incubation for 2 h at 37 °C
- Transformation in E. coli (5 μl)
