Digested DNA migration pattern on agarose gel for southern blot

Digested DNA migration pattern on agarose gel for southern blot

Southern blot digested DNA abnormal migration pattern on agarose gel:

I get pretty good quality DNA from mouse tails (using phenol/cia) to use
for Southen blots. I digest the DNA in a large volume and then
precipiate it in NaCl/ethanol. When I run the digested DNA for the blot,
it doesn't run in an even smear, but in a shape which is sort of hollow
in the middle and gets narrower toward the bottom of the gel. Ideas
we've had are that there is too much DNA in the well, too much salt in
the DNA, or SDS in the sample, and we've tried to fix these, but still
haven't gotten the samples to migrate correctly.
thanks.

DNA runs faster in the absence of ethidium bromide than in its presence.
I think that "hollow" lanes are caused by all the EtBr being bound up
and exhausted by the DNA in the middle of the lane, so that DNA there
runs faster. Diffusion in from the area between lanes would bring more
ethidium bromide to the sides of the lane, so these regions would see
more ethidium and the DNA would run slower. Lots of RNA, which would run
the fastest and also sop up ethidium bromide, certainly wouldn't help.
Possible cures:
1) less DNA per lane
2) raise ethidium bromide concentration in buffer to 1 ug per ml (may
help but not cure).
3) treat with RNase to break up RNA into small fragments that would tend
not to absorb so much ethidium bromide.
4) Run the gel without any ethidium bromide at all (I have never tried
this, but it certainly should work and would more or less test the
theory.) Note that the mobility of DNA compared to tracking dyes would
be increased--run times or voltage might have to be changed if you have
some standard set of conditions.

Last update 02-Aug-2007, Rating n/a of 1 votes.

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By Kulbir on 02-Aug-2007

I have been doing southerns in wheat. I will also suggest to run gel without any ethidium bromide. You can sytain afterwards withEtBr to see it. Also make sure that Digestion is good. Enzyme should be sufficient to digest the DNA.
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Technique / Molecular Biology / DNA analysis techniques / DNA southern blot

Digested DNA migration pattern on agarose gel for southern blot