Pfu

Peter Friedhoff at chemie.bio.uni-giessen.de
Mon Jan 16 11:08:56 EST 1995


In article <D_henrique-1601950951180001 at mac030005.ox.icnet.uk>, 
D_henrique at icrf.icnet.uk says...
>
>Dear netters
>Can I have the experienced oppinion of someone that uses routinely Pfu
>polymerase for amplifyng products for cloning. Is it worth to prevent
>mistakes in the PCR? I want to use the PCR product for cloning and then
>expression in E.coli and am afraid of mistakes that can result in
>truncated proteins.
>Thanks
>Domingos

You should use in any event a proofreading polymerase (Pfu or Vent or 
other). When using Taq you have to use very special condition to reduce 
the error rate. Usually I use Pfu-DNA polymerase for site-directed 
mutagensis cloning a 700 bp nuclease geneand only very few clones (about 
1 out of 20) had a point mutation. If you have very long PCR products (> 
5000 bp) you may include Taq in the reaction (Barnes et al. 1994, PNAS, 
91, 2216-2220)
Hope that helps for a decision. 

Peter




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