KOD HiFi DNA Polymerase - (Feb/26/2007 )
i am not familiar with KOD Hifi DNA polymerase although i have purchased one:)
the yield is very low - 4-5 times lower- than that of regular Taq. I guess a decrease in yield is something expected for highly proofreading polymerases. But i need to ask whether this much decrease is expected or not. Do I need to increse the extension time to give the polymerase enough time to proofread or I just go ahead with the times I have already been giving to regular taq.
Thanks in advance.
I have no experience with KOD hifi, but in my experience I do not get low yields with pfx (which if I'm not mistaken is the same enzyme, just another provider). I would optimise your PCR with KOD and play with annealing temps, optimal Mg is lower for pfx (so I assume also for KOD), this might influence your annealing temps.
An increase in extension time is not necessary, KOD is highly processive (it's actually faster than taq, check this article http://www.ncbi.nlm.nih.gov/entrez/query.f...=pubmed_docsum)
thank you very much for your prompt response vairus,
i will optimise the pcr again then.
One more thing, I forgot to mention, but maybe you should check your buffer composition, as Invitrogen (provider of pfx) advises users to use doulbe concentrated buffer for doing PCR with pfx, maybe KOD has the same issues? I have been more succesful indeed with double concentrated buffer, but I don't know if the KOD buffer would double concentrated vs pfx-buffer.