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.
|Plasmid DNA (50 ng)
|primer 1 (10 μM, 125 ng)
|primer 2 (10 μM, 125 ng)
|10x reaction buffer
- 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 10 μl of the Dpn I-treated DNA from each control and sample reaction into separate 50-μl aliquots of BL21 cells
No colonies were seen the next day.