GENOMIC SOUTHERN: help with DNAseI Hypersensitivity (please!!!!!) (:

Heidi Moss hmmoss at MAIL.MED.CORNELL.EDU
Mon Aug 26 13:38:50 EST 1996


hello all bionetters!!

i have been attempting to perform a DNAseI Hypersensititvity assay from 
Kit225 (T cell line) nuclei to determine the hypersensitve sights in my 
promoter. i have the assay going fairly well in regards to nuclei 
isolation, DNase treatment, quality of genomic DNA, etc. HOWEVER, my 
problem with obtaining quality, interpretable data occurs in the VERY 
last simple stage of the genomic Southern blot. I observe the expected 
decreasing intensity of my expected major band with increasing 
amounts of DNAseI. HOWEVER, the minor "hypersensitive" bands are there, 
but so difficult to see over the background DNA smear which is non-specifically 
hybridizing (the membrane itself is beautifully clean). I have tried 
EVERYTHING: adjusting washes, temperature, etc... i either lose the 
signal all together for the hypersensitive bands, or get too much 
background. ANY ADVICE, EVEN ON GENOMIC SOUTHERNS IN GENERAL WOULD BE 
APPRECIATED. 

	this is what i have been doing:
nuclei isolated---> treated with 0, 3, 5, 7 U DNaseI---> genomic DNA isolated

-- Digested 40micrograms of DNA (I tried 10micrograms, but was advised 
for THIS type of assay to use more...) with XbaI/SacI
-- Ran agarose gel--> BLOTTED WITH 0.4M NaOH (should I stay away from 
alkali blotting??)
-- Hybridized with a 1kb 10-9cpm probe in FBI buffer (basically, a PEG 
buffer) using HSDNA as a non-specific carrier at 65 degrees ON
-- Washed at RT at first-->65 degrees going from 2xSSC/0.1%SDS-->0.5xSSC

HOW CAN I GET RID OF THE NON-SPECIFIC SMEAR WITHOUT LOSING MY FAINT 
HYPERSENSITIVE SIGNAL??	

thanks all

--Heidi




More information about the Methods mailing list