ethidium bromide: add to gel before pouring or stain afterwards

Dom Spinella dspinella at chugaibio.com
Mon Jun 29 09:26:51 EST 1998


> y lab collegues and I have been debating this for a while now...
> 
> To detect RFLP's, we currently add ethidum-bromide to a melted, high
> resolution gel, then re-nuke it so to pour an even gel. I, however, think
> this is a highly mutagenic way to do things, particularly since the gel is
> volatile, the ethidium following with the vapour... I would prefer to stain
> the gel afterwards, following by a destaining bath in water, of which I have
> noted to work perfectly fine...
> 
> I have visited other labs doing gel electrophoresis, and most seem to stain
> the gels after running.. not prior to running into the gel before pouring...
> this is sometimes hard, especially when using large gels (minimum of 30
> wells)...
> 
> Please, end our constant in-fighting! Does anyone else add the ethidium prior
> to nuking and pouring? or do most of you RFLP'ers etc stain the gel after
> running?

I've been adding EtBr to the pre-nuked agarose for years now (although I
used to stain the gels after running). The absolute mobility of DNA does
slow when run in EtBr gels, but the relative mobility doesn't -- so it
doesn't affect the interpretation of the results at all if you're using
a ladder for size comparisons (and who doesn't?). The big advantage of
having EtBr in the gel is that you can run it with the rig on top of a
UV box and you can visualize the bands in real time to be sure that you
have achieved optimal resolution.  Its also faster and the bands are
brighter (nice for visualizing small quantities of DNA).  As you point
out, post-run staining will work fine as well but I've never had the
slightest problem with incorporating EtBr directly into the agarose.
IMHO, if its faster and more convenient, and gives equivalent results,
why not use it?  BTW the slowing of migration rate is not due to the
fact that EtBr moves "backwards" (i.e. towards the cathode) as someone
suggested.  That "reverse migration" is simply due to electroendosmosis
-- the movement of water through the gel following salt and carrying the
EtBr with it.  EEO happens whether or not there is EtBr in the gel...
Cheers. Dom Spinella



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