Northern - prehybr.

SEPPO KAIJALAINEN MMETT kaijalainen at viikki.Helsinki.FI
Wed Jun 23 00:58:35 EST 1993


My problem is the backround signal when I have total-RNA (5-10 ug,UV 260nm) 
on the nylon (Boehr. +++). The RNA is denatured with Glyoxal/DMSO. According 
to my results with 1X oligo-dT selectedRNAs on the filters (nice bands in 15 
min after adding the colour sol'n with DIG-labeled RNA-probes) the target 
should be high copy-No mRNAs (several cDNA-clones as probes). With the 
totRNAs the whole area  where you could think RNA to exist, is purple, with 
the sense AND the antisense-probes. Sometimes the smaller rRNA-band remains 
white like the rest of the filter. The pattern is similar with 32P-random-
primed ds-probe (4X 10 to 8 cpm/ug, 500 000 cpm/ml ofhyrd.sol'n) although it 
takes O/N to appear. I've used two different prehyb-hyb-buffers;The one 
suggested by Boehringer and the other: 0.1 M Phosphate pH 7.2, 7% SDS, 5mM 
EDTA,50% deion. Formamide, 50 ug yeast-RNA/ml in all sol,ns, 68o C, 
prehybridization 6-20 h. The high stringency wash is 40 mM P-buffer, 1% SDS,
5 mM EDTA, 2X 1/2 h 68oC. I find preparation and quantitation ofpolyA-RNA 
frustrating, and would like to use totals instead. If there is anyone who 
recognizes the problem, and could advise -help !

                           S.K.



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