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April 25, 2013
- QuickChange Site Directed Mutagenesis
- Use 50 ng of dsDNA template and 125 ng of each primer. Template strands are about 5kb for H2B-GFP and 7kb for LOV.
| Reagents |
H2B |
LOV
|
| Plasmid DNA (50 ng) |
0.4 μL |
0.2
|
| primer 1 (10 μM, 125 ng) |
1.7 |
1.7
|
| primer 2 (10 μM, 125 ng) |
1.7 |
1.7
|
| 10x reaction buffer |
5.0 |
5.0
|
| dNTP mix |
1.0 |
1.0
|
| dH2O |
40.2 |
40.4
|
| Total |
50.0 |
50.0
|
- Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to each control and sample reaction
- Thermal cycling
- 95°C/30 sec
- [95°C/30 sec; 55°C/1 min; 68°C/7 min (1 min/kb plasmid length)]x18
- 4°C, ∞
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