Pfu DNA polymerase

Yasuyuki Kurihara kurihara at mac.bio.bsk.ynu.ac.jp
Tue Nov 21 19:49:55 EST 1995


In article <1995Nov16.144708.1 at sara.cc.utu.fi>, elebae at sara.cc.utu.fi
wrote:

> 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
non-specific!
> I know all my other reagents are O.K, because I get really good
products with
> the DYNAZYME polymerase. I have tried different concentrations of
glycerol,
> different temperatures, different amounts of template, but nothing
seems to
> help. I would really appreciate some tips, because the enzyme is too
> expensive to just  make tens of reactions/trials...I would also be
interested
> in hearing about the accuracy of this enzyme. 
> Thanks a lot!
> Elena

Hellow Helena!
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.
Good luck!

-- 
Yasuyuki Kurihara
Yokohama National University
e-mail:kurihara at mac.bio.bsk.ynu.ac.jp



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