band focusing on DGGE

harem at BSCR.UGA.EDU harem at BSCR.UGA.EDU
Thu Jan 26 11:15:36 EST 1995


Hello DGGE practitioners,

I am trying to break into the DGGE world of allele separation, but
I'm having some difficulties.  The quick question, which I will
elaborate upon below, is: will these smears on my 11 hour gel become
progressively more focused when I run a longer gel?

I have a 300 bp AT-rich fragment with a 40 bp GC-clamp attached.  It
has a flat computer-generated melting curve for the entire length of
300 bp, followed by the high melting domain of the GC-clamp (at the
My perpendicular gel worked fine, indicating that at 55 degrees, the
steep transition occurs between 40 and 50 % denaturants.  So I ran
a time-series experiment on a parallel gel, 2 hr intervals, 11 hrs
total.  The short run times have large fuzzy bands with no evidence
that different alleles (or homo/heteroduplexes) are being resolved.
The long run times all proceeded to nearly the same place in the
gel (i.e. the distance between adjacent lanes diminished with longer
run times, as expected), but the bands completely diffused into a
very light smear.

Will this smear focus into discrete bands, or am I investing in one
of those rare fragments that just wont focus for reasons unknown?
Thank you in advance to anybody out there who can shed light on this
question.  My GC-clamp primer is unpurified, and part of my fear is
that this is diminishing or preventing proper resolution of a focused
product.

Cheers, Matt Hare
University of Georgia
Harem at bscr.uga.edu



More information about the Methods mailing list