Silver staining of aDNA in agarose gels

Paul N Hengen pnh at ncifcrf.gov
Thu Apr 17 11:16:00 EST 1997


Philip L. Carl (plc at med.unc.edu) wrote:

> We are initiating some differential display experiments, and as several 
> sources have suggested that the traditional denaturing acrylamide gels in fact 
> yield many "artifactual" bands I have been doing some preliminary experiments 
> using FMC Metaphor agarose for displaying the bands.  The gels are much 
> stronger than low concentration acrylamide gels, and I suspect that another 
> advantage of agarose is better recovery of bands for reamplification.  The 
> problem is detecting the bands.  I'd like to avoid radioactivity if possible 
> for the usual reasons.  We have tried SYBR Green I, but like some others find 
> that it is not very much more sensitive than EB-perhaps as much as a factor of 
> 10 fold (i.e. a few ng), but probably less in our hands-certainly not the 60 
> pg claimed in the tech literature.  We've also tried silver staining using the 
> method of Gottlieb and Chavko Anal. Biochem. 165: 33-37 (1987) which  claims a 
> lower limit of detection of 2.5 ng-a claim we believe reasonable.  Trouble is 
> that this is probably about 1/100 as sensitive as staining acrylamdide with 
> silver.  So my question is, does anyone know of a better silver stain for 
> agarose gels? 

Phil:

I think you could try a simple press together technique for transferring some
material onto a nylon membrane for more sensitive detection methods like
chemilumenscence and save the gel. Not all of the DNA is transferred out of
the gel and you can go back to it for isolation of fragments later if necessary.
You could detect the DNA by biotinylating it, either before with nick translation,
PCR with 5' biotin on the primers, etc., or after using psoralen-biotin and
one of the Streptavidin-AP detection kits available. I find this to be very
sensitive, and really not much more work than silver staining. You can
probably even cut a slice from the membrane for PCR amplification, although
I've not tried it.

--
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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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