high background on northern blots

chai_z at wehi.edu.au chai_z at wehi.edu.au
Thu Jul 15 19:08:42 EST 1993


Path: wehi.edu.au!chai_z
From: chai_z at wehi.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: high background on northern blots
Message-ID: <1993Jul16.100806.1 at wehi.edu.au>
Date: 16 Jul 93 10:08:06 +1000
References: <CA7s46.Fpq at ns1.nodak.edu>
Organization: Walter & Eliza Hall Institute
Lines: 33

In article <CA7s46.Fpq at ns1.nodak.edu>, borovkov at plains (Alex Borovkov) writes:
> 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.

I had the same problem in Southern with certain batch of 32P (no explanation).
But I found if you reused the probe it would be fine in the second round of 
hybridization. So, I suggest that if you could not find the cause you might
try to absorb your probe using a blank membrane following the same procedure of
prehybridization, hybridization and then boil the probe (ds DNA probe) before
adding it to the hybridization with your Northern blot.

Good Luck.

Zhonglin Chai



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