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.
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).
at 11:51 AM