Southern blot nightmare

Anita Gould anita at accord.cco.caltech.edu
Fri Nov 11 06:05:42 EST 1994


In article <1994Nov7.101220.5277 at ucbeh> converrl at ucbeh.san.uc.edu writes:

	I was writing regarding a problem I have with stripping a
	Southern Blot from a Magnacharge NT membrane:

   1. After probing this genomic Southern with a 2kb nick translated
   probe (100 microliters of 3*10^5 cpm/microliter probe), my exposure
   shows that probe is hybidizing all over the blot in a random
   pattern (it looks like an inkblot test!). My boss told me that this
   type of background comes from not filtering the probe through a .45
   micron Polysulfone acrodisc syringe filter prior to hybridization.

   2. I have never encountered background like this before and do not
   make a habit of filtering my pobes.

   3. After Stripping this filter and reprobing it, I got the exact
   same "inkblot" pattern as before. My boss seems to feel that this
   is protein &/or other contamination that will not come off no
   matter how extensively I strip. My stipping conditions are: 70
   degrees Centigrade, .05 N NaOH, 0.1X SSC, 1% SDS. I change this 2
   times and wash 30 min. each time (we have one of those rotating
   hybridization ovens).

	My questions are the following:

   1. Is the assesment that I did not filter my probe the correct one
   for the source of all this background?

   2. Is there any way I can strip the "junk" off this blot without
   removing the DNA?

			 E-mail any helpful suggestions!!

			 Richard

I have had exactly the same problem.  I haven't got it nailed down
completely, but here is the current state of my knowledge (or lack
thereof :-) ) If anybody out there has a definitive solution, there
are now at least 2 of us here who would love to hear it!  In the
meantime, Richard:

I agree with your boss that filtering the hyb and prehyb solns just
before use may be what's necessary.  (I don't know of any rationale
for filtering the probe itself -- unless anybody else chimes in with
one, I would just filter the hyb before adding probe.  Even 10%
dextran sulfate solutions will go thru a filter, much to my surprise.)
I think that the blotchy or punctate crud comes from probe sticking to
aggregates or precipitates of SDS &/or blocking agent -- either the
protein or gooey hi-MW DNA (as suggested by another poster).  In fact,
there have been times when I have seen a visible precipitate anytime I
let my prehyb or hyb cool below hyb temperature.  (This was using 37
deg for a 50% formamide/4X SSPE/0.1% SDS prehyb with yeast tRNA and
nonfat dry milk as the blocking agents.)  The problem seems to be
worse with tRNA/milk, but I have also gotten it with
Denhardt's/sheared DNA.  I really suspect that the culprit is the SDS,
because a) the ppt goes back into soln with gentle warming (50 deg),
b) a similar ppt also forms in the stock bottle of prehyb (sans
blocking agents) if it has not yet been filtered, & c) I have had this
problem even with Amersham's Rapid-hyb, which I recently started
using, and which supposedly doesn't contain any protein or nucleic
acid blocking agents. (Rapid-hyb lets you do hybridizations in 1-2.5
hrs.  No affiliation!  Haven't gotten all the bugs worked out yet, as
you can see, but hope to soon -- *if* I can manage to debug without
knowing what's in their proprietary formula.)  Anyway, filtering all
solutions just before use (and being fanatical about keeping your
membrane clean and dust out of everything) gets rid of nucleation
sites, and I have seen it make a dramatic difference.  Keeping the
prehyb & hyb from cooling (even for 5 or 10 min.) once they are
applied to the membrane mayhe
prehyb & hyb from cooling (even for 5 or 10 min.) once they are
applied to the membrane may also help.

The other thing that I have tried that seems to help is very gently
massaging the entire surface of the membrane (wearing 2 pairs of
gloves) after it is transferred to the first wash buffer (& maybe the
2nd as well).  The idea is to dislodge any tiny particles of crud that
are sticking to the membrane, & won't come off with just the normal
wash action.

Like you, I have found that this crud does not come off with any
normal stripping protocol.  But I was able to get most (though not
all) of the counts off with a Proteinase K treatment of the membrane:
100 ug/ml Prot K, 1% SDS in TE pH 8, 3 hr @ 50-55 deg.  The remaining
counts were unfortunately refractory to further treatment (maybe they
were due to SDS particle residue from the SDS-protein clumps, or to a
nucleic acid component), so this is not advertised to be a cure, but
it might help.

Best of luck!  Please summarize any replies to the net, or at least
forward them to me.
--

                                   -Anita Gould
                                    anita at cco.caltech.edu



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