Team:Tuebingen/Notebook/Protocols/mutagenesis PCR
From 2014.igem.org
(Difference between revisions)
Line 51: | Line 51: | ||
<tr> | <tr> | ||
<td style="text-align: center">to 50 µL</td> | <td style="text-align: center">to 50 µL</td> | ||
- | <td> | + | <td>H<sub>2</sub>O</td> |
</tr> | </tr> | ||
</table> | </table> |
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
- 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)