In article <1995Nov16.144708.1 at sara.cc.utu.fi>, elebae at sara.cc.utu.fi
> Hi there!
> Could somebody give me some advice concerning the Stratagene Pfu DNA
> polymerase(recombinant version)? I have tried quite many different PCR
> conditions, but still I don't get ANY product at all, not even
> I know all my other reagents are O.K, because I get really good
> the DYNAZYME polymerase. I have tried different concentrations of
> different temperatures, different amounts of template, but nothing
> help. I would really appreciate some tips, because the enzyme is too
> expensive to just make tens of reactions/trials...I would also be
> in hearing about the accuracy of this enzyme.
> Thanks a lot!
In my experience, amplification by native pfu DNA polymerase is better
than that of cloned one. But please note that the purity of the primer
used in this reaction is critical. We experienced no amplification by pfu
polymerase when tr-off primers were used. So, tr-on and OPC or gel
purified primers are essential. If you use such primers, the other basic
parameter is annealing temperature. Or you had better use pwo DNA
polymerase(Boeringer). Performance of this enzyme is similar with pfu.
The accuracy of these enzyme is very good. We have cloned many DNA clones
from cDNA library by 30-40 cycles PCR with pfu. But no mistake was
observed untill now.
Yokohama National University
e-mail:kurihara at mac.bio.bsk.ynu.ac.jp