mouse tail DNA for Southern blot
mantei at cell.biol.ethz.ch
Wed Apr 26 09:11:21 EST 2000
In article <39060104.BC24C98A at aecom.yu.edu>, alami <alami at aecom.yu.edu>
>Does anybody know how to fix this problem?:
>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.
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.
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.
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
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