help with missing contact footprinting needed!
The Wagner Lab
ewlab at uci.edu
Tue Nov 7 16:03:48 EST 1995
I am trying to do some missing contact footprinting (limited methylation,
followed by gel shift, then cleavage rxns). Can anyone suggest some
guidelines about incubation times for limited methylation based on DNA
size? I have been given conflicting advice on the G+A rxn (formic acid)
and T+C rxn (hydrazine). Once source says that the longer the DNA, the
shorter the incubation time, another says the exact opposite. I have
used per rxn 200ng single-end labeled 25mer probe. For G+A I have used
25ul formic acid, incubated at RT for 60min. For T+C I have used 30ul
hydrazine, incubated at RT for 20 min. The cleavage rxns show some bands
which sort of appear to be differentially sensitive in comparing the
bound vs. free species, but the results are less than convincing. I
would greatly appreciate any advice/personal experiences. Thanks!
--
___________________________________
Matthew Petroski
mdpetros at uci.edu
(714)824-6953
Molecular Biology and Biochemistry
University of California, Irvine
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