Pfu DNA polymerase

Jody K. Hirsh jkh141 at nwu.edu
Mon Nov 20 16:53:43 EST 1995


In article <1995Nov16.144708.1 at sara.cc.utu.fi>, elebae at sara.cc.utu.fi 
says...
>
>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
-- 
Dear Elena:
Not only do you need to use the buffer that comes with the Pfu, but you 
need to extend the time for DNA synthesis (Pfu is less processive than 
Taq) AND also lower the annealing temperature about 5 degrees or so 
because of the salt conc. difference between the one you probably 
calculated using Taq's conditions. I had the same problem, and when I 
made these changes (suggested by the Stratagene Tech Rep--call them!!!),
It worked!

Jody K. Hirsh
Northwestern University, Chicago, IL.   USA
jkh141 at nwu.edu




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