extracting dna from acrylamide gels

[Liz Parks]

Jeffrey,
Acrylamide will inhibit PCR, EtBr will not.  I have had a great deal of
success using a method from Biotechniques (Nov. 1994 Vol. 17, No. 5, pgs
914-921).  Basically, cut out the band of interest from the dried or fresh
gel.  Incubate it at 95C for 20 minutes in 100 ul of 10 mM Tris-HCl, 50 mM
KCl, 1.5 mM MgCl2, 0.1 % Triton-X, pH 9.0.  Use 1 ul in your subsequent PCR
reaction.  In my experience, this works better than water.

Regards,
Liz

Jeffrey Markert wrote:

> I'm looking for advice on extracting DNA bands from acrylamide gels.  My
> goal is to recover enough DNA for a few PCR reactions. The acrylamide gels
> are stained in EtBr and were made with FMC's Long Ranger mix.
>
> I've been using the ammonium acetate elution protocol from Maniatis, but
> I'm not finding it to be very reliable in my hands and it would be nice to
> find a procedure that doesn't require an o/n incubation.
>
> I'm wondering whether there's a simpler and quicker way that will work for
> PCR.  Do acrylamide or EtBr inhibit PCR?  Could I just elute the gel slice
> in water to avoid having to get rid of the ammonium acetate?  Could I just
> freeze (or boil) the gel slice?  Any thoughts or anecdotes are welcome.
>
> ---

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Liz Parks
North Carolina State University
Department of Plant Pathology
Raleigh, NC  27695
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