joining PCR fragments into a single product

Mathias Holpert mholper at gwdg.de
Fri Sep 28 08:50:46 EST 2001



Peter Ashby schrieb:

> 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.

... an overlap of 30 bp also works for fragments up to 3,5 kb- but then the
efficiency is going down.

Mathias




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