Low Fluorescence DNA Gels

Bryan L. Ford fordb at bcc.orst.edu
Mon Aug 18 01:53:43 EST 1997


Eugenie L. Harris wrote:

 
> I concur with Frank that most people use far too much EtBr.  But I'm
> going to go one better and say all this staining and destraining
> business is a waste of time.  We use Metaphor agarose for microsatellite
> typing and save a lot of money by reusing the gels up to 10 X in a
> simple load and run, load and run strategy.  When we run a gel for the
> first time we usually put EtBr in the gel before pouring it.  After that
> we just put a mimimal amount of EtBr in the bottom tank.  The DNA
> migrates to the anode.  The ethidium migrates to the cathode.  They
> meet, the bands are stained, the photo is taken, the buffer is changed
> and the gel is loaded and run again.  Since we use minimal EtBr,
> background is very low.  The EtBr concentration in the bottom tank is
> roughly 0.03 ng/ml.  (I'm not quite sure on that amount since I can't
> remember the volume in the lower tank and am guessing it.)
> 



Hello Jean:

Thanks for the useful suggestions. It appears that running one's low
concentration EtBr from the (+) chamber should be a good method to
further facilitate "no destaining" on polyacrylamides even though your
mentioned use is with Metaphor. I suspect that this approach may prevent
my concern about the "elution losses" (see my reply to Frank O. Fackel).
One can hardly do better than effectively avoiding both staining and
destaining altogether. Have you used this method with polyacrylamide as
well? 

I like the idea of repeatedly using the same gel-- I think our present
vertical minigel apparatus would not allow the required reassembly as
currently configured, unless one had non-fluorescent and UV transparent
plates, to allow UV excitation and photography directly through the
glass. Are you using a horizontal slab apparatus? Or is there a vertical
unit that allows for facile multiple photographs/runs?

-Bryan




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