EtBr in agarose gels vs. EtBr staining later

Andy Bates bates at liv.ac.le
Mon Feb 27 10:35:40 EST 1995


In article
<Pine.A32.3.91.950222182822.74692A-100000 at rs5.tcs.tulane.edu>
Brick Ayola <abrick at mailhost.tcs.tulane.edu> writes:

> 
> Regarding EtBr staining of nucleic acids:
> Can one simply add EtBr to the loading dye (as with some RNA gel 
> protocols)? Or will this significantly disturb the migration of DNA 
> super-coils and such?

The behaviour of supercoiled DNA on an agarose gel is a complex area. 
However, in general, if you are interested in the state of
supercoiling, then you have to stain afterwards.  Almost any amount of
EtBr in the sample or the gel will result in the closed-circular DNA
running with positive writhe on the gel, that is with high mobility
relative to nicked, or open-circular DNA.  In other words different
levels of supercoiling will not be resolvable.  If this is confusing,
then get back to me privately if you like.

If you don't care about such subtleties, then adding EtBr to the sample
will probably work to some extent, but the longer you run the gel, the
fainter the bands will get, because the EtBr will tend to
electrophorese in the opposite direction to the DNA, and hence will
gradually be stripped off the DNA.  

Putting EtBr in the gel works well, but the same stripping out will
occur once the samples get beyond the boundary of EtBr which moves up
the gel as the samples move down.  This method also gives you a lot of
background, if the sample is in the region of gel containing the EtBr.

For the prettiest results, I would always stain afterwards, although
you can destain a gel which was run with EtBr in it to reduce the
background.

Best of luck,

Andy Bates

____________________________________________________________
Dr. Andrew D. Bates             Tel:   (+44) (0)151 794 4322
Department of Biochemistry      Fax:   (+44) (0)151 794 4349
University of Liverpool         Email: bates at liv.ac.uk
PO Box 147
Liverpool
L69 3BX, UK



More information about the Methods mailing list