Pfu polymerase in PCR

test user at mail.ncku.edu.tw
Wed Feb 12 18:04:46 EST 1997


> >Bob Steinberg <RSTEINBE at ETOWAH.UOKHSC.EDU> wrote:
> >>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

We have met this problem two years  ago. We couldn't get any PCR product
with buffer #1. Then, we made the buffer with same components and pH
value by ourselve, and figured out the problem. We have done an
experiment that one reaction run PCR rxn with buffer supplied and the
other with home-made buffer. Only the home-made buffer got PCR product.
We didn't know what wrong about the buffer it supplied. Now, we use our
own buffer and it works well. Hope this help.

Chung-pei Ou
Institute of Biochemistry
NCKU



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