mouse tail DNA for Southern blot

Ned Mantei 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> 
wrote:

>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. 
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.

-- 
Ned Mantei
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland




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