Boiling ethidium bromide gel

Vladimir Svetlov svetlov at oncology.wisc.edu
Wed Apr 29 11:04:28 EST 1998


In article <REPLACE-2904980901440001 at macbb2212b.unil.ch>,
REPLACE at lbm.unil.ch (Stephan) wrote:

> > Unbound EtBr, you mean? But that's good. Let it. Since it's travelling to
> > the catode from the well (and not from across the entire gel) it's not
> > gonna affect the brightness of the field at all. Normally you'd have to
> > wait a while before the bottom part of your gel is cleared from the EtBr.
> > On the other hand, bound to DNA EtBr travels with it to the anode - these
> > intercalating dyes don't dissociate that easy - so that yer DNA remains
> > nice and shiny in the UV. So I don't think that this metaphysical argument
> > really stands up. 
> 
> Mmmm... I wouldn't be so sure about the dissociation. 

Well, if you would be so kind as to provide the dissociation constant and
the hal-life for EtBr-DNA complex in TBE, we could actually leave the
metaphysical ground. Oh, shit, I forgot "Mmmm". 

> EtBr DOES dissociate
> from the DNA, and quite well I would say (have you ever done EtBr-CsCl
> extractions?). 

Never in my life. I did EtBr extractions by n-butanol or using Dowex column
from CsCl solutions. Neither extracting agent is a component of my
electrophoretic protocol. Mmmm. 

 > If the concentration of EtBr is constant in the gel and in
 > the buffer, an equilibrium is reached.

Even in recirculating buffer there always be a gradient pattern of EtBr in
the electric field and ... Stop, even  better - please right out a kinetic
analysis of EtBr-DNA dissociation for a compact band of a finite volume
instead of infinitely dilute and momentarily equilibrating solution. 

 >The dissociated molecules will move
 > toward the cathode, while the DNA will move toward the anode. If there's
 > no EtBr to replace the one that has just dissociated and moved towards the
 > cathode, your bands will appear more and more faint, eventually until you
 > can't see them anymore.

Same thing would "eventually" happen in the gel with EtBr added to it,
would it not? Are you going to such a great length to justify the primal
urge to flood everything including the running buffer with EtBr? 

It's too bad that Polya's work on plausible reasoning is not a required
reading for biology students. Apart from being a truism, this deliberation
sadly lacks any indication to what condition one have to establish for the
gel run for this dissociation to actually become a problem for nucleic
acids detection with UV.  
 
> There's no need of methaphysics to understand that.

Tha'sss right, my lad. What needed though is some knowledge of kinetics.
Unless you are willing to entertain my questions above I think we can cease
this pointless discussion of why things should not work the way they do.
Frankly speaking, if the need comes for that kinda mental exercise I'd
rather take it up with Zeno (no offense, man). 

Have fun,
V.

-- 
Vladimir Svetlov, Ph. D.
McArdle Lab for Cancer Research
Dept. Oncology
UW-Madison
1400 University Ave.
Madison, WI 53706



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