ligations in LMP agarose
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)
> >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
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