PCR and polymerase.

bruce w. ritchings bwr at icbr.ifas.ufl.edu
Fri Dec 22 16:53:40 EST 1995


Dale Levitzke wrote:
> 
> Hello netters,  I am currently using exo-pfu polymerase for the
> amplification of a G-C rich PCR.  Has anyone had any experiance in using
> this enzyme?  Has anyone had any experiance in the amplification of G-C
> rich templates and if so what was your enzyme of choice ?
> Would greatly appreciate any input.
> 
> Ta
> 
> Dale Levitzke, Biomolecular Research Facility, Newcastle.Dear Dale,
We have routinely used pfu polymerase in our pcr reactions
with Pseudomonas DNA which is 67% GC.  Although it is easier
to get the amplification product with Taq polymerase, Taq makes more
errors compared to pfu. Also, pfu makes blunt end products
which can be cloned.  The only differences that we do in our PCR 
reactions are that we add 10% DMSO (dimethyl sulfoxide) and decrease the 
annealing temperature in the PCR reactions (e.g. if we use 55 degree C if 
using Taq, we will use 47 or 50 with pfu).  Give it a try.  Good 
luck!Dale Levitzke wrote:
> 
> Hello netters,  I am currently using exo-pfu polymerase for the
> amplification of a G-C rich PCR.  Has anyone had any experiance in using
> this enzyme?  Has anyone had any experiance in the amplification of G-C
> rich templates and if so what was your enzyme of choice ?
> Would greatly appreciate any input.
> 
> Ta
> 
> Dale Levitzke, Biomolecular Research Facility, Newcastle.



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