Site-directed mutagenesis - nucleotides insertion (Feb/20/2009 )
I used Stratagen UltraPfu and DpnI digestion for site-directed mutagenesis. I would like to make insertion of my clone. How many nucleotides have you ever tried for insertion in one mutagenesis? I have tried 12bp. Does anybody try longer one?
You can insert an entire gene to any nucleotide position using this automated molecular cloning method described by Chen and Ruffner. You'll have to sign up to view the graphs, which you do in order to understand how to it work and how to design the primers. Once you learn this elegant method all your future site-directed mutagenesis and cloning work would be a piece of cake. BTW, pfu pol is not the best for large plasmid and other long read DNA polymerases work better. Good luck!