PCR optimization

Michael Sullivan mlsulliv at wisc.edu
Tue Jan 17 10:54:30 EST 2006


On Jan 17, 2006, at 4:53 AM, Duncan Clark wrote:

> Historians believe that in newspost  
> <1137469560.315751.95860 at g49g2000cwa.googlegroups.com> on Mon, 16  
> Jan 2006, vagos <el36 at le.ac.uk> penned the following literary  
> masterpiece:
>> Have you got any ideas on how I could improve on this?
>
> 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.
>
> Duncan

Duncan,

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.

Mike
---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)



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