ligations in LMP agarose

mazella mazella at naxos.unice.fr
Tue Apr 19 06:58:22 EST 1994


>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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Georges GAUDRIAULT
Institut de pharmacologie moleculaire et cellulaire
660, route des lucioles
06560 valbonne
FRANCE
e-mail :mazella at naxos.unice.fr
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