high background on northern blots
antje at RT1.MENZIES.SU.EDU.AU
Fri Jul 16 13:51:53 EST 1993
> >>> RCPT To:<reagents at net.bio.net>
> From: antje (Antje Haase)
> Message-Id: <9307160305.AA26218 at rt1.menzies.su.edu.au>
> Subject: Re: high background on northern blots
> To: borovkov at plains (Alex Borovkov)
> Date: Fri, 16 Jul 93 12:05:36 CST
> Cc: methods, and, reagents at net.bio.net
> In-Reply-To: <9307151630.AA16374 at net.bio.net>; from "Alex Borovkov" at Jul 15, 93 4:21 pm
> X-Mailer: ELM [version 2.3 PL11]
> > 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.
your problem is most likely too high probe concentration or too much
radioactivity. We were having this problem occasionally. The dirty blots were
done in exactly the same way ( using identical membrane and reagents) as
blots that came out fine, the only difference being the probe. Once we
reduced the amount of probe/label to half they were clean and beautiful
Menzies School of Health Research
Casuarina, NT 0811
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