Southern blots - cause of Denhardt's spots

danielg at uncmed.med.unc.edu danielg at uncmed.med.unc.edu
Fri Sep 21 09:52:41 EST 1990


Recently I posted a note concerning various problems with Southern blot
autoradiography, and had a few responses.  My main problem is "Denhardt's
spots".  The following suggestions have come in, and I will include my
comments.  Am hoping for more suggestions, explanations, and encouragement
-there *has* to be a way to eliminate these @#!*&%@! spots.

1. Agarose stuck to the filter after transfer:

	It was suggested that I manually, lightly rub the surface of the
	filter after transfer, before baking, to remove agarose particles
	that would cause spots on the autorad.  I objected to this at
	first b/c the spots were so round (looked like bubbles, not blobs
	of squished agarose), but the writer suggested that the pieces
	were so small that I couldn't see their true shape.  However, I
	discarded this theory b/c after we stripped the filter and
	reprobed, the spots showed up in diffent places on the filter.

2. Bubbles in pre-hyb/hyb:

	This was a good suggestion, or at least, it lined up with our
	thinking. However, we carefully pre-hybed and hybed, squishing the
	solution around in the bag every so often, to move any bubbles
	that might be stationary.  The spots did not even diminish.  Blah.

3. Precipitation of Denhardt's onto the filter:

	Some have suggested that when you transfer the filter from hyb to
	wash solution, that the Dextran sulfate, or some other component
	(nonfat dried milk, if your solution contains that, but don't
	quote me, I'm so tired of figuring out what does what in the hyb
	solution, I need a vacation!) precipitates onto the filter (due to
	temperature change?  Doesn't formamide freeze near RT?) and must
	be manually removed by rubbing membrane after first wash.  We are
	trying this now.

4. Poor washing:

	We tried washing the filter longer, but band intensity decreases
	as much as the noise, so the probe is stuck just as well.

	One writer suggested that when you dilute the Dehardt's by placing
	the filter in the first wash, the probe is able to stick to the
	blot where ever it likes.  He suggested that the first wash be
	vigourous.  Any comments.

I suppose I could do a little more thinking before I post, so if I have
overlooked something obvious, please have mercy!  All help welcome, rescue
us from this terrible malady, I see spot before my eyes!

dan
___________________________________________________________________________
 **** The shallow man has *** |  Do not be overly righteous, nor overly
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