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slowing down fast bands

Ludwig Lab-UCSC niflab at biology.ucsc.edu
Fri Aug 13 17:52:31 EST 1993

nntp at acsu.buffalo.edu writes:

>Dear Net:

>	I am interested in sequencing/footprinting on relatively small
>DNA sequences.  A 20% denaturing PAGE gel will accomodate almost the entire
>sequence, but the smallest fragments usually get very spread-out and the
>very smallest  zip right off the end of the gel.  One person suggested 
>pouring a gel of variable thickness to create an electrical field gradient.
>Large pieces would migrate faster, small pieces would migrate slower, relative
>to their mobilities in a standard "flat" gel.  This makes sense to me, and
>I have seen examples of such gels.  After checking searching the archives 
>for this group, I have seen nothing on this topic except a reference to 
>thermal gradient gels which may yield similar results.  
>welch at sc3101.med.buffalo.edu

>John Welch

I have used buffer gradient gels for many years on all my seq gels.
The spreading of the bands at the bottom is eliminated quite simply.
No reason not to use them for oligos on 20% gels.  The DNA gets down in
the high Tris region and slows down.  The gradient is mixed in a pipette
by drawing a few bubbles through the interface between two solutions (using
sucrose to make the lower denser and some BPBlue to make it visible.)  
See Biggin et al. PNAS (USA) 80:3963-3965, 1983.

	Dr. Robert Kuhn
	Sinsheimer Laboratories
	University of California
	Santa Cruz, CA 95064  USA
	ph:  408-459-2728
	fax:        -3139 
	email: rkuhn at orchid.ucsc.edu

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