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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