I'm fixing several cDNA clones with point mutations using
site directed mutagenesis. I'm using the Stratagene
QuickChange protocol but using KOD HiFi polymerase
rather than Pfu Ultra because I can get almost 100%
product every time with KOD. After PCR I do a DpnI digest.
All the clones I sequence have the point mutation fixed.
However, a high percentage of the clones have the mutated
region duplicated--exact copy of the primers used. It
looks like a tandem region.
I've seen only a little talk of this happening and why, but
no real solutions to the problem. I synthesize the oligos
myself and desalt them prior to use. Of the 4 clones I pick
for each "fixit" reaction, all of them will have this duplication.
KOD is very fast and for a 6kb plasmid, my extension time
is only 40 seconds. After 23 cycles I get tons of product.
However, I see the same problem with Pfu, too.
Any ideas? Thanks, Paul