Posted 24 September 2010 - 10:37 AM
I don't think it really matters. You are going to be changing two bases no matter which you choose. Changing these two bases is not a factor in site-directed mutagenesis. Heck, I've deleted up to 108 bases in one construct with the overlapping primers and made up to 9 point mutations with about 20 base changes in another, all in one pcr. It's more important to make sure your primers are designed well and you get the right pcr conditions. Granted, to do the deletion I had to raise the pH and lower the MgCl2. I have a set of pcr optimization buffers where the pH, MgCl2 and KCl are all varied. Let me know if you run into problems and I will send you the recipe for these buffers. I've always managed to get a pcr to work in at least one of the conditions.