SDM large plasmid - (Jul/30/2005 )
Anyone ever tried Site directed mutagenesis on large plasmids?
I'm most worried about the polymerase to use. (Technique used will be like quickchange site directed, but just buy my own enzymes/competent cells and not the kit).
I have done SDM on plasmids ~10kB very successfully using a Quikchange based approach. I have been using Pfu Turbo, due to its high fidelity. Taq and other polymerases apparently generate too many errors that effect your plasmid. Furthermore, from what I have read, Pfu seems to be the 'gold-standard' polymerase to use for site-direct-mutagenesis. In my ignorant honours days ( ), I used Taq, and that gave me more colonies on my plates following heat shock of the PCR samples (which were positive mutants). However, after reading more about the different polymerase, and what ppl use in high-impact papers, I decided to begin using Pfu Turbo.