pwo polymaerase

Arnoud van Vliet avvliet at knoware.nl
Fri Aug 18 19:58:57 EST 2000


> I have tried to use PWO (proofreading) DNA polymerase for PCR cloning.
> With 'normal' Taq polymerase I get a nice one band PCR product. When I
> use PWO with similar PCR conditions I get a smear on my agarose gel. I
> have tried increasing the Mg-concentration, increasing the annealing
> temp., changing both the enzyme and template concentration, the number
> of cycles and elongation time, with and without DMSO. So probably most
> common problems have been eliminated. Has anyone any other ideas???? I
> am out of any...

Well, if Taq works fine, try to clone the Taq-made fragment. Depending on
the purpose of your experiment, that might be good enough to use. Or do you
absolutely require it to be done by Pwo? Maybe try another proofreading
polymerase, or a taq/proofreading enzyme mixture like Expand from Roche (no
affiliations).

hope this helps
Arnoud







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