Pfu polymerase in PCR

Wed Feb 5 20:14:13 EST 1997

Does anyone have suggestions for making recalcitrant primer/template 
combinations work with Pfu polymerase. We are trying to amplify a short 
(about 120-bp) segment with very high fidelity-- eventually from genomic 
DNA, but presently from a plasmid template. We have no problem getting 
the correct product with Taq or ULTma polymerases, but get nothing with 
Pfu, even though we have dropped the annealing temperature by more than 
10 degrees from what works with the other polymerases and have 
phosphorothioate linkages for the three nucleotides at the 3'-ends of our 
primers. Extension times at 72 degrees C have been 2.5 min, which should 
be more than generous for the short piece we are trying to amplify. We 
have tried two lots each of Pfu polymerase, Pfu buffer, and dNTPs. Has 
anyone looked at varying salts and/or magnesium with Pfu?

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