ligations in LMP agarose
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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