Northern blot trouble

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Wed Nov 13 15:54:10 EST 1996


In article <56c5ku$h78 at merkurius.lu.se>, Fredrik Kamme <fkhero at biogen.wblab.lu.se> writes:
> Hi folks,
> I've got a northern blot problem that goes like this:
> I'm storing total RNA + 2vol eth at -20°, which I then ppt and run
> formaldehyde gels, that look pretty nice. Everything has worked nice so
> far, but now when I hybridize with cDNA probes or oligos, I get a fat
> spot on my autoradiograms just at the well. Why? Is this RNA that never
> dissolved? No obvious ppt's in my load sol's and nothing obvious when I
> stain the gel with eth. bromide. The protocol has worked previously, but
> now it doesn't. Is there a ppt formed over time when RNA is stored like
> this, that is more difficult to dissove? I'm flabbergasted...:-(
> 
> Thanks for any help, a bewildered Freddy

Two possibilities come to mind, 1) your RNA isn't dissolving properly
which can be remedied by dissolving your RNA in 5ul of DEPC H2O 65 C
for 5 min before adding the loading buffer.  2) DNA contamination, Ive
seen hot "Bands" at the tops of my northerns at times which
disappeared when I increased the time on ice to let the phenol get rid
of the HMW genomic DNA (or adding a second phenol extraction, which I
now routinely do) during the RNA isolation.  What tipped me off is
that I was extracting multiple samples and the ones I did first (i.e.
the longest with phenol ccl3 on ice) had the least amount of the upper
band and it progerssively increased in subsequent samples.  Just a
thought.  Good luck

					Regards, 	SVEN




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