joining PCR fragments into a single product
p.r.ashby at dundee.MAPS.ac.uk
Thu Sep 27 11:37:45 EST 2001
In article <f0bf934e.0109270731.29af788b at posting.google.com>,
ayboro at my-deja.com (Alex) wrote:
> Hi all,
> I'm wondering if anybody can help me with a reference. I remember an
> article (I think at was in Biotechniques) on a simple polymerase based
> method that allows to generate a single fragment from two PCR
> generated overlapping halfs. Combining products of 5' and 5' RACE into
> one contiguous DNA fragment without restriction/ligation has been used
> as an example.
> Do anybody remember the article and can provide me with the reference.
I have used this method for in vitro mutagenesis. You mix the two
overlapping fragments together in equimolar ratio (after purification)
and perform 3-5 cycles of pcr with no primers. Then you add primers for
the extreme ends of the combined fragment and amplify. The idea is that
the overlap of the fragments act as primers and generate a few molecules
of full length which you can then amplify. Sorry I can't give you a
reference. In my experience an overlap of 20-30bp works for fragments up
Wellcome Trust Biocentre
University of Dundee
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