Friday, June 12, 2009

All your amplification needs

In a nicely-timed post, Damn Good Technician just happened to recommend a polymerase for amplification from genomic DNA, KOD polymerase from EMD. I had never heard of this polymerase, but I have recently become quite familiar with the fact that Phusion polymerase (Pfu fused to a dsDNA binding protein) from NEB/Finnzyme really sucks for amplification from gDNA. I know it is the enzyme itself that sucks because (after several rounds of troubleshooting that, naturally, assumed other things were the problem) I compared Phusion to regular old Taq -- Taq did them all, Phusion did none (oddly, Phusion worked on one when the template was a BAC, so I guess there is something about the complexity of the gDNA, or at least the relatively rarity of primer/template complexes that Phusion can't handle).

Since it is a Friday and I am impatient, I'm not going to wait for my KOD to arrive sometime next week, so I'm going to try a few rounds of Taq amplfication and use that as substrate for the Phusion, hoping that once Taq 'gets it started' Phusion can take over from there.