joining PCR fragments into a single product

Peter Ashby 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 
to 500bp.

Peter

-- 
Peter Ashby
Wellcome Trust Biocentre
University of Dundee
Dundee, Scotland
Reverse the spam and remove to email me.




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