Inverse PCR troubleshooting
I have been trying the inverse PCR method to change a single amino acid, so i designed back to back primers, with the forward primer having the mutation. I then did the PCR, phosphorylation, and ligation protocol (overnight). When I transform by electroporation, there usually is no colonies. When there are colonies (about 2), there are no mutations for the most part. And when there is the desired mutation, there are huge insertions (200 bp) that seem to come from no where. I really dont know what to make of my results, and i would very much appreciate the help. Also if anyone has more troubleshooting techniques, I would be glad to hear.