Site-directed mutagenesis problem - Mutations under oligo (Aug/29/2007 )
I've run into a problem. I setup a site-directed mutagenesis reaction using Stratagene's QuikChange SDM kit. I transformed their competent cells (after DPNI digestion) & got ~10 colonies/plate. I sequenced 10 colonies & all contained the original wild-type base that I tried to correct. I double-checked my HPLC-purified primers to ensure the sequences are correct for making a mutation and they are. Stratagene suggested re-amplifying plasmid with a 68deg annealing/extension step. I tried this & after DPNI digestion, got >100 colonies/plate. Great! However, I sequenced (now) 24 colonies and found a strange phenomenon: some of the clones now contain the mutation, but all clones have deletions, duplications, or other mutations under the site of the annealing primers. In total, according to DNA sequencing (which is clean), I got 0 clones that contain the mutation and have no other mutations.
I wonder if anyone has run into this before or could offer a potential solution. I've "racked my brain" but don't think of anything that would have caused this. I've used the sequencing primers before on other clones, and they work fine.
try to add 5% DMSO while amplifying the gene. This could help solve the problem.
good Luck !!!
Scolix: if you want to I would be happy to her an explanation to that.