high background on northern blots

Antje Haase 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.
> > 
> > 
 Hi Alex,
 
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
 
 Good luck
 
 Antje
 
 
 _________________________________________________________________________
 Antje Haase
 Menzies School of Health Research
 Casuarina, NT 0811
 Australia
 _________________________________________________________________________
 
 
 
 
















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