Slanted Bands on Agarose Gels

botn056 at csc.canterbury.ac.nz botn056 at csc.canterbury.ac.nz
Sun Jun 5 17:15:41 EST 1994


In article <tbrown-020694120627 at eagle.health.ufl.edu>, tbrown at nervm.nerdc.ufl.edu (Thomas A. Brown) writes:
> In article <1994Jun1.144723.28647 at news.unige.ch>, mike at divsun.unige.ch
> (Mike Morris) wrote:
> 
>> In article 206 at usenet.ucs.indiana.edu, wfischer at bio.indiana.edu (Will Fischer) writes:
>> *Christopher W Botka (cwbotka at MERCURY.SFSU.EDU) wrote:
>> *: Has anyone experienced bands(DNA) that run slanted on an agarose gel? -
>> *: i.e. the bottom of the EtBr stained frag. of DNA seems to be migrating
>> *: faster than the top. 
>> *
>> *  Seems the culprit is different buffer conc. between gel and
>> *running buffer, generally due to volume loss while dissolving the agarose.
>> *
Yeah, I only ever seem to get this problem if I make the gel too far in
advance, and it dries out.  The you almost have to tilt the transilluminator to
figure out what happened.  Now, if I know a gel will be sitting for a while I
cover it with running buffer.





>> 	I have always believed that it was due to a TEMPERATURE gradient in the gel, not buffer concentration. I avoid it by running gels more slowly, or 
>> in the cold room.

I _accidentally_ ran a gel late one night when all I REALLY wanted to do was go
home.  I had forgotten to freeze the gel box (filled with 50% ethylene glycol),
and the agarose actually remelted.  Once it had re-solidified, it was one of
the sharpest gels I have ever run.  No band slanting at all.
I (nearly) always run a gel at ambient, and rarely have this problem.

> 

> We've found that it is often due to differential EtBr concentration.  If
We don't put EtBr in our gels.


On of the other postings on this thread cited evaporation of the buffer when
melting the agarose. IF I ever melt the agarose in a microwave (I'm an old
fashioned guy who prefers a heating block) I ABSOLUTELY make sure that I mark
the liquid level in the flask, so I can top up with distilled H2O before I
pour.  I don't need to do this on a heating block, as the mix doesn't have to
boil as long before the agarose is all melted - strange but true!  (you heat
for longer, but boil for less time).


Glenn Manning

Manning at botn.canterbury.ac.nz

These opinions are mine and mine alone.  Not yours, mine.  Not your neighbours,
mine.  Alone.



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