Isotope Incorporation? HELP

ez001427 at dale.ucdavis.edu ez001427 at dale.ucdavis.edu
Mon Aug 15 16:06:43 EST 1994


Dear Netters-

	I'm having an odd problem with the creation and use of a 
tritiated substrate DNA.  Maybe one of you can shed some light on the 
problem.  I have used tritium-labeled dimethyl sulfate to add labeled 
methyl groups to a large quantity (20 mg) of pBLuescript II KS+ plasmid 
DNA.  As per the protocol, after letting the reaction run, I precipitated 
the DNA away from the unincorporated label, washed the pellet, 
resuspended it and dialyzed it several times, until the dialysis buffer 
was not so hot.  I then froze the DNA in aliquots for use.

	PROBLEM: The label should be in the form of methylated DNA bases, 
and I am trying to study the release of these labeled bases from the DNA 
backbone.  Unfortunately, when I take a sample of the DNA, and do an 
ethanol precipitation, I get a large percentage of the counts in the 
supernatant fluid! This happens when I use 2 to 5 volumes of 100% 
ethanol, whether or not salt (Na or Ammon. Acetate) is present.  I've 
done this several times, as well as trying a re-precipitation step 
first.  In all cases, the lion's share of the counts don't precipitate, 
despite the fact that I see a pellet and have dialyzed away essentially 
all of the unincorporated or degraded label.

	I have also tried to use spin-filters (Millipore Ultrafree-MC), 
but under the conditions I was using, the counts went right through the 
300Kda membrane (the plasmid is 1.8 million daltons).  I think that my 
microfuge doesn't spin slowly enough, and I was putting about 7Kgs on 
membranes rated at 2-5 kgs, so no wonder there were problems there.

	Any suggestions or comments would be highly appreciated.  My 
objective is to be able to detect the released methylated bases by LSC, 
but so far, I can't seem to keep the label where I want it.

Thanks in advance,
Marc Goldstein:  magoldstein at ucdavis.edu
UCD Plant Biology




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