Sequencing gel combs (leakage)

Brian Foley brianf at med.uvm.edu
Mon Mar 28 23:59:29 EST 1994


the End (jgraham at bronze.ucs.indiana.edu) wrote:
: Hello comrads,

: Has anyone figured out why sequencing gels will leak on many occasions
: allowing loaded samples to flow into adjacent spaces ? I use a "shark's
: tooth" comb and more than half of the time find movemnet of the laoded
: sample between the plate and the comb.

	Bummer.  It will make your sequence unreadable, bands in all
four lanes plus...  I hate it when that happens...

	Are you clamping the top spacer in while the gel polymerizes?

	Do you use the same comb as a flat spacer that you use for
		sharks teeth?  I even make sure I keep the same
		side on the left end (rotate it about its long
		axis to go from flat well-former to teeth down
		multi-lane well-former) in case the plastic
		from which it was made had variations in
		thickness.
	
	Can you buy a bar to clamp the top of the plates into
		your gel running apparatus?  Some vertical units
		allow the glass plates to bow a bit unless you
		buy a bar clamp for the top.
	

: I often allow my gels to polymerize merely several hours while others
: swear that overnight is necessary. 

	One hour is plenty.  At least with my ammonium persulfate and
		TEMED formulation it is.

: This may lead to a soft edge at the 
: top perhaps. Otherwize swelling and seperation of the gel from the 
: plate may be occurring during the pre-run.

	I doubt it.  Does the load sneak under the teeth, or does it
		leak between the glass and the teeth?  If it is 
		sneaking under the tips, just push the comb down in
		more (but not too far or it will distort the lane edge).

: Any tips greatly appreciated. At this point I would just a soon load 
: into regular analytical wells due to this problem.

	What?  Don't you get a perverse pleasure from watching hours
		work slowly go down the drain because of a dirty
		rotten little technical detail?  If not, how have
		you managed to get this far?

: thanks,

: Jim

: E-mail copies if possible


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