extracting dna from acrylamide gels

Mr JN Goulding jgouldin at hgmp.mrc.ac.uk
Fri Nov 3 07:15:16 EST 2000


Hi,

I've done some amplification from Acrylamide gels; both by boiling the gel
in water for 5 mins then adding 10ul, and also just adding the gel
straight into the PCR reaction (they were 100 ul pcr reactions) and got
good results both ways. Usually now I just add the Acrylamide into the pcr
reaction as it's less hassle and I've not had a problem doing that. Hope
it helps,

Jon Goulding

On Thu, 2 Nov 2000, Liz Parks wrote:

> 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.
> >
> > ---
> 
> --
> 
> ~~~~~~~~~~~~~~~~~~~~~
> Liz Parks
> North Carolina State University
> Department of Plant Pathology
> Raleigh, NC  27695
> ~~~~~~~~~~~~~~~~~~~~~
> 
> 
> 






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