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, ∞
- DpnI Digest (gets rid of methylated template DNA)
- H2B mut rxn
- H2B control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
- LOV mut rxn
- LOV control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
- Add 1 μL DpnI enzyme to each sample
- Gently and thoroughly mix each reaction, spin down in a microcentrifuge for 1 minute, and immediately incubate at 37°C for 1 hour to digest the parental supercoiled dsDNA
- Transform 1 μl of the Dpn I-treated DNA from each control and sample reaction into separate 50-μl aliquots of BL21 cells
|
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