high background on northern blots

Alex Borovkov borovkov at plains
Thu Jul 15 11:21:41 EST 1993


Hi netusers.
Can anybody tell me what I am doing wrong. I blot the formaldehyde/agarose
gels after few washes in water in 20xSSC. After UV crosslinking and washing
in 5-6xSSC the membrane prehybridizes in 0.5M Na-phosphate pH7.0, 7% SDS and
0.1% BSA for few hours at 65C and hybridizes to randomly 32P labelled probe
in same solution. After then the membrane washes in 2xSSC > 0.1 SSC.
As you can see the procedure does not have anything unusual. I use it many
times in the pass without any problem. Now I always have very high background
which I can't rid of. I use Nybond-N and Nybond-N+ (any suggestion which one
is better?). I tried hybridization with formamide, transfer in 7.5mN NaOH and a
runing in regular 1xTAE buffer but nothing works. The background is not even
and does not trace the RNA runs but split all over the membrane.
A southern at same condition and with same probe works fine. 
Any suggestion and comments will be greatly appreciated.
--
Dr. Alex Borovkov,                   | Please excuse me for my english.
Plant Pathology Dept.,               | My russian is getting worse too
North Dakota State University        | and soon I will know nothing 
Fargo, ND 58105                      | but few dirty words.



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