ligations in LMP agarose

Darren Martin Martin at gene.unp.ac.za
Tue Apr 19 04:26:05 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.

  



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