Quick Change (QuikChange) Mutagenesis (From Hahn Lab Protocols):
Notes on oligo design for QuikChange
1. Order oligos (both directions) that have the desired change plus 22 bases or so on either side. 2. Set up separate primer extension reactions for each oligo, consisting of: 1. 5 ul 10X pfu buffer (supplied with pfu turbo enzyme) 2. 1 ul 10 uM primer 3. .1-.2ug plasmid template (1 ul miniprep) 4. 5 ul 2 mM dNTP mix (or 1 ul 10 mM) 5. 1 ul Pfu turbo polymerase 6. H2O to 50 ul (37 ul for 5 ul dNTP and 1 ul template) 3. Run through the following program 1. 94oC 30 sec 2. 95oC 30 sec 3. 55oC 1 min 4. 68oC, 2 min/kb up to 10 KB plasmid 5. Repeat 2-4 for a total of 4 cycles, then hold at 4oC. 4. Combine 25 ul from the two extension reactions above. 5. Add another 1 ul of pfu polymerase. 6. Run the same program, but cycle for 18 times instead of 4. 7. Take 25 ul of the reaction, and add .5uL (10 U) DpnI enzyme. 8. Incubate for 1 hour at 37oC 9. Transform and plate.
If this method proves unsuccessful, and DMSO doesn't help, use inverse PCR with phusion instead (design non-overlapping oligos, one of which has the desired change. After pcring, phosphorylate the purified product, then blunt-end ligate). As per Julius, it's actually more efficient to just phosporylate the primers before doing the pcr. Basic idea here