Mutagenesis troubles - (Jun/11/2010 )
I did mutagenesis once in the past without any problems, but I can't get it to work now. I am using the Stratagene quickchange protocol (I haven't bought the kit but am just using all the required ingredients).
I am trying to introduce a single mutation, and I think my problem may be that the region I am targeting is very GC rich. The oligos I have got are about 75% GC.
I have tried two different annealing temperatures (60 and 55 degrees), 5% DMSO, decreasing the primer concentration and increasing the template concentration without any progress.
Any help would be greatly appreciated. I read somewhere else that trying an annealing temp of 68 might help.
did you use the Finn enzymes (Dna polymerase) from NEB they work great with mutagenesis i use it for my latest mutation.
they are called Phire Finnzymes call NEB ask for free sample and try