PCR optimization

Austin P. So (Hae-Jin) nobody at nowhere.com
Tue Jan 17 20:00:26 EST 2006

Michael Sullivan wrote:
> On Jan 17, 2006, at 4:53 AM, Duncan Clark wrote:
>> I would try using a left hand primer that ends with the A mismatch  at 
>> it's 3' end. Preferably use that with a proofreading polymerase  i.e. 
>> Pfu or a.n.other.

> I would think a non-proofreading polymerase should be used in this  
> case. Otherwise, the polymerase might remove the 3' terminal primer A  
> prior to extension. Unless my reasoning here is wrong. In any case,  iI 
> agree that the best way to discriminate between the two plasmids  is by 
> having the mismatch at the terminal nucleotide of the primer.

People have introduced 3'-terminal phosphorothioate linkages into the 
primer which would prevent editing of the 3'-end by the proof-reading 
polymerase (I think it was with Vent, although in principle it should 
work just as well with Pfu).



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