pcr from ligation

Peter Ashby p.r.ashby at dundee.MAPS.ac.uk
Thu Oct 28 09:34:49 EST 1999


In article <38185AC8.861E8A0E at laplace.csb.yale.edu>, Mark Edward Bowen
<mb at laplace.csb.yale.edu> wrote:

> I want to ligate two dna fragments together an amplify the resultant
> product for subcloning.    Does anyone have any tips on getting this to
> work well.  Is it necessary to clean up the ligation before PCR?  Should
> I keep the ligation equimolar or doesn't it matter?  Will  self-
> ligation products products prove probelmatic?

When I last did this I just put a small amount of the ligation straight
into the pcr mix and it worked fine. I would expect that eqimolar would be
the way to go.
If your fragments can self ligate then you will have problems. You can
check for this by doing control pcr's which contain only one oligo. If
your fragments are significantly different in size you should be able to
work out which band is the one you want and gel purify it for subcloning.

Peter

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



More information about the Methods mailing list