Drying agarose gels

mkbarnhart at uh.edu mkbarnhart at uh.edu
Wed Mar 24 12:09:08 EST 1999


Hi,

You have another alternative.  Try running a combination acrylamide/agarose
gel. The composition is 3%-5% acrylamide and 0.5% agarose.  These gels have
been used to do gel shifts on splicing complexes.  I've also used them to do
gel shifts on relatively small RNAs and large protein complexes.

To pour the gel, make up your acrylamide in 1/2 the total volume and 1/2
total volume of 1% agarose.  Melt the agarose normally and allow to cool to
about 50 degrees.  Add your Ammonium persulfate, the agarose and then quickly
pour the gel. The heat from the agarose will cause polymerization fairly
quickly. Either run your gel slowly or in a cold room so the agarose doesn't
melt.

You can expose these gels wet.  If your reaction is hot enough you can expose
them for a couple of hours at 4 degrees C or if needed you can expose them o/n
at -80C.

If you have any questions, email me.
Michael Barnhart
Laboratory Supervisor
Connective Tissue Physiology Lab
University of Houston



In article <36F6969E.7A27 at ucdavis.edu>,
  James Kami <jakami at ucdavis.edu> wrote:
> A method I used many years ago came from a Hoffer techniques pamphlet.
> Essentially, the gel has first denatured with NaOH as if preparing for a
> southern blot, then dried onto standard filter paper using a vacuum gel
> drier for one hour with no heat followed by one hour with low heat, less
> than 55 degrees so as not to melt the gel. After drying the gel has
> rehydrated in DI H2O and floated free of the filter paper. The result
> was a thin, leathery material that was remarkably tough. It could be
> used for hybridization just as a Southern blot on filter paper, although
> stripping had to be done chemically rather than by heat. The great
> advantage was that I could use small probes (20-40 bp) and had NO
> background. The gel could be reused many times and still gave extremely
> clean RFLPs. I used probes up to 500 bp with no problem.
> 	Depending upon the size of your protein, I wonder if you might not be
> able to incubate a dried DNA gel with a protein solution and detect
> protein binding useing an antibody or other method. My understanding is
> that, unless the DNA-protein binding is of very high affinity, gel shift
> sometimes doesn't work. Anyway, it was just a thought.
>
> Good Luck
>
> Jim Kami PhD.
> Blue Rose Biotech
> Davis, California 95616
> Dr. Fernando Rodriguez-Pascual wrote:
> >
> > I will be very grateful if someone can give me some tips to dry agarose
> > gels. The purpose is to perform a kind of electrophoretic mobility shift
> > assay to look the possible interaction between a 560 bp radioactive
> > transcript and a purified protein. For this transcript size I should use
> > agarose instead PAGE, but I was told that the drying of agarose gels is
> > not easy.
> >
> > Thank you in advance
> >
> > --
> > #############################################
> > Dr. Fernando Rodríguez Pascual
> > Department of Pharmacology
> > Johannes Gutenberg University
> > Obere Zahlbacher Str. 67
> > D-55101 Mainz, Germany
> > Tf. +49 (6131) 17 68 36 or 17 32 45
> > Fax. +49 (6131) 17 66 11
> > email: frodrigu at mail.uni-mainz.de
> > #############################################
>

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