Recombinant Circle PCR

Don Jackson djackson at welchlink.welch.jhu.edu
Tue Mar 14 12:45:48 EST 1995


In article <3jq82f$9i6 at dns1.NMSU.Edu>, dkim at nmsu.edu (Daniel Kim) wrote:

> 
> Hello:
> 
> I was looking into directionally cloning PCR products into a plasmid, and
> stumbled on an article in PCR Methods and Applications  3:S141-S148 (1994)
> "PCR Mutagenesis and Recombination in Vivo".  This article describes a
> method for combining PCR products in vivo by transfecting linear DNA
> fragments with homologous ends into E coli.....(deleted)...
> 
> Before I start designing primers, I'd like to know if anyone has
> experience with this process.  Is it easier than more conventional
> methods?  Does it really work as advertised in these papers?
> 
> Thanks.
> 
> Daniel Kim
> dkim at verdi.nmsu.edu
Daniel
Our lab has used this technique extensively for directed mutagenesis with
excellent success, but from your posting, I gather you're more interested
in using it to subclone.  I usually try to have 20-30 bases of overlap on
each end of the linear DNA's; one of the Biotechniques articles has (I
think) some data on how length of overlap affects recombination efficiency,
but I don't think a 4-6 bp overlap (i.e., a restriction site) would work
particularly well.  This means you either need longer primers or have to be
apble to settle for lower efficiency.  I also find that the "cleanliness"
of your PCR template is important; even small amounts of intact plasmid DNA
can make life unpleasant.
I still subclone the old fashioned way.
-- 
Don Jackson
Dept. of Molecular Biology and Genetics
Johns Hopkins University School of Medicine
djackson at welchlink.welch.jhu.edu



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