Gel smiling problem

Bruce Windoffer brucew at connectnet.com
Mon Sep 15 11:05:28 EST 1997


Bryan L. Ford wrote:
> 
> pradeep at boseinst.ernet.in wrote:
> >
> > Dear netters:
> >
> > I am repeatedly encountering a problem which I heard is known
> > as "smiling" of PAGE (denat) gels run at high temp (50-60 C).
> > Outer lanes in the gel show a distinct curvature toward the outer
> > edges. So far as I can remember, this is usually caused by
> > non-uniform heat distribution/dissipation. However, I
> > could not find any discussion on this phenomenon in the Current
> > 
> > Short Protocols on Molecular Biology (Condensed version of the
> > "Red book", we do not have the full book here) or in Maniatis.
> > The book "Gel Electrophoresis of Nucleic Acids" (IRL)
> > is also not much help, it mentions this effect but does not
> > discuss. I would really appreciate references/papers on this
> > effect. Specifically, I am also interested in knowing
> >
> > 1. Can "smiling" be caused by any factor other than heat?
> > 2. Is it possible that acrylamide/bis contained impurities
> >  (if they are not very good grade) leading to unequal distribution
> >  of heat or distortions in the electric field ?
> > 3. Is it possible that glass plates lose uniformity after repeated
> >  use and may contribute to this effect ?
> >
> 
> Friend:
> 
> My advice would be to stick with the heat hypothesis, especially in view
> of the temperature at which you are running your gels. This problem is
> easily overcome using a good sequencing apparatus, that is one with some
> means of distributing the heat with great uniformity. But I am guessing
> that you may already be using a sequencing apparatus, and in that case I
> would advise making sure that the heat distribution system (buffer or
> aluminum plate) is properly functioning. Also I would ask why you are
> running your denaturing gel above 50 degrees C? Provided you have
> adequate denaturant (Urea or Urea+formamide or ?) concentration in your
> gel and provided you heat denature in formamide containing loading
> buffer (or alkali denature) your templates immediately prior to loading,
> you should be able to keep virtually all strand/strand associations at a
> very low to zero level even with as low as a 40 degree running
> temperature (optimum being 40 to 50).
> 
> As for references, I believe you may find some discussion of this
> looking the the full size "Red Book", especially under "DNA Sequencing",
> but smiling is so routinely a predictable symptom of too much heat that
> it is often overlooked in descriptions of electrophoresis.
> 
> In case the above recommendations are not enough and with sincere
> apologies for the lecture I will give you my brief explanation of
> heat-generated "smiling": You apparently already know that temperature
> has a profound effect on DNA (or marker dye) mobility, so that modest
> temperature lowering will slow progress through a gel. The edges of your
> gel plates are cooler than the center for any of several reasons that
> are to some extent characteristic of your particular electrophoresis
> apparatus. In general terms the center of the gel is being resistively
> heated from all sides and can cool mainly only from the faces of the
> glass plates. However the edges are effectively heated only from one
> side and they usually have a free edge or edge clamp that effectively
> increases the area for heat dissipation as well. Hence the edges are
> almost always cooler-- and thus the mobility of DNA etc. is lower at the
> edges.
> 
> In most sequencing apparatus one usually avoids loading wells near the
> edge of the gel to eliminate or minimize the distorted results.
> 
> I hope this is helpful;
> 
> Sincerely,
> Bryan


Bryan's very informative "lecture" regarding the causes of edge smiling
effects on vertical electrophoresis systems is correct.  Our company
makes systems which correct for this, but I will refrain from "selling"
on Usenet.

However, our experience differs from Bryan's with respect to running
conditions for complete denaturation of some DNA.  With a high enough
G-C content, gels need to be run at or above 50 degrees to remove
secondary structure effects on electrophoretic mobility.  This is
necessary even after heat denaturing the templates and running with
sufficient denaturant (urea) in the gel.

55 or even 60 degrees may be necessary for complete elimination of
compressions. Running at 45-50 degrees is just not sufficient for these
DNA templates.  Of course, most systems are not designed for
temperatures this high.

Regards,
--Bruce Windoffer
http://www.genomyx.com



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