We use iproof polymerase from Bio-Rad and a pair of outward primers to PCR the whole plasmid, then phosphorylate the ends, ligate the PCR product into circular plasmids, and then transform.
It used to work quite efficiently. If the template plasmid has been efficiently removed in DpnI digestion, almost every colony gives the right construct. Lately we observed random deletions at the mutagenesis site at quite high frequency. These are either single nucleotide deletion from either end of the primer, or a deletion of 5-20 nucleotide stretch.
Anyone knows how to fix it? Was it because we switched from phusion to iproof, poor primer quality, poor PCR extension?
Thanks a lot!
They are both proof-reading polymerases and as such have 3´→ 5´ exonuclease activity. I'm not familiar with iproof, but adding the polymerase right at the end of the reaction is important to avoid this exonuclease activity chewing up your primers.
So if you have changed the order of addition of reaction components or stopped preparing on ice (iproof protocol suggests it is thermostabilised) then that could be a reason.
Or perhaps it is a new student (ie slow pipetter) that has the problem?
Or perhaps your primers have been freeze/thawed too often?
Could test this by doing a reaction with Phusion and see were you get....
Would love to hear the result of your trouble shooting!
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