pcr from ligation

John R. McQuiston zje8 at cdc.gov
Thu Oct 28 10:36:37 EST 1999

We have done this but we had to dilute the ligation mix 1:100 to get good 
PCR.  Anything higher gave us multiple bands.


In article <p.r.ashby-2810991534490001 at paimac.bioch.dundee.ac.uk>, 
p.r.ashby at dundee.MAPS.ac.uk says...
>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 Ashby
>Wellcome Trust Building
>University of Dundee
>Dundee, Scotland
>Reverse the spam and remove to email me.

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