Sequencing phenomenon

[Brian Foley]

Andrea M Skantar (skantar at unity.ncsu.edu) wrote:
: Perhaps this has been covered before, if so, pardon the repetition. Does 
: anyone know what causes a wierd phenomenon in sequencing gels where the 
: tracking dye in the center goes SLOWER than on the edges?  I've heard 
: about the dye going slower at the edges and faster in the center being 
: due to non-uniform heating of the gel, but this is a new one on me.  It 
: gives the gel like a fish-eye effect at the bottom.  I've gotten readable 
: sequence from such gels but it sure looks wierd.  Any ideas?
: Andrea

	Have you heard of wedge gels?  Wedge gels use a spacer that is 
thicker at the bottom than at the top, so those small DNA fragments that 
would otherwise run off the bottom of a normal gel after a long run slow 
down and will be retained on the gel.  This happens because there is less 
current per cross-sectional area flowing through the thick part of the gel.
This could account for the center of a normal gel migrating slower than 
the edges.  If you pour a gel and polymerize it in a vertical or slanted 
position the plates will bow out ever so slightly to make the center of 
the gel thicker than the edges.
	Glass plates are not as rigid as one would think.  They are 
actually quite easy to bend.  To get a perfectly uniform gel you need to 
lay it nearly flat during polymerization.

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
********************************************************************