ligations in LMP agarose

Bernard Heymann bheymann at bragg.bio.purdue.edu
Tue Apr 19 13:14:56 EST 1994


In article <2p0h0u$8mu at mserv1.dl.ac.uk>, mazella at naxos.unice.fr (mazella)
wrote:

> >I am ligating 125 ng pUC18 (SmaI digested and partially end filled) with +-
> >50 ng insert DNA (+- 2 Kb Sau3AI digestion fragments that are also partially 
> >end filled) in a 60 ul volume of LMP agarose.  The Buffer in the agarose is 
> >a mixture of 0.5X TBE and 1X T4 DNA ligase buffer.  Following ligation (
> >using 6 weis units of T4 DNA ligase incubated at 4 degrees overnight) and 
> >use of the product to transform E.coli (using an apparently excellent 
> >transformation protocol), I only get a small  number 
> >of transformants (50-600 with +-60-80% containing insert).  I was wondering 
> >whether anybody had any "high frequency of ligation tips" when doing 
> >ligations in LMP agarose.
> 
> >Any help will be much appreciated.
> 
> I think you shouldn't use Borate in all buffers you use for these
> experiments because it inhibits the ligase.
> 
> 
> Hope it will help you
> Georges
> 



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