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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