Overlap extension PCR

Stein Tore Solem steins at nfh.uit.no
Mon Sep 13 05:28:42 EST 2004


In article <BD64D367.9DC%eescott at ku.edu>, eescott at ku.edu (Emily Scott) 
wrote:

> I am hoping users have some suggestions to assist with a problem I am having
> with overlap extension PCR/SOE (splicing by overlap extension) and multimer
> formation.
> 
> ---



Dear Emily

This might work (It does for me..): Use 50 ng each of the gel-purified 
templates. Cycle at least 8-10x, then add your outer primers (15 pmol) 
and do 25-30 additional cycles. To avoid frameshifts and/or 
pointmutations in the junctions of the two fragments, I believe it is 
important to us a proofreading polymerase in all appplications, but in 
particular during the first PCR when the SOE-templates are generated.


Hope this helps,

Stein Tore



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