Creepy sequencing gels

scarr at kean.ucs.mun.ca scarr at kean.ucs.mun.ca
Sat Nov 6 14:15:05 EST 1993


In article <tshin.752470128 at husc8>, tshin at husc8.harvard.edu (Tae Shin) writes:
> Hi there;
> 	I recently had a problem with running sequencing gels, one that I
> have never before encountered in my three years of lab work.  After
> polymerization and removing the shark-tooth comb (which was
> inverted to create the top edge), I usually pre-run the gel before loading
> it.  In a relatively short period of time, the top edge of the gel had
> moved upwards and about a 1/4" had actually extruded out from between the
> plates!  After talking with the Bio-Rad tech support, he suggested that
> gels have a natural tendency to do this and it is the friction between the
> gel and the glass which immobilizes it.  Hence, the reason for the
> creeping was due to over-siliconizing the plates.
> 
> 	I concurred with him, since I do apply Sigmacote to both plates. 
> After stripping one plate, the problem has disappeared and the gels have
> run without a hitch.
> 
> 	Now, ignoring that fact that I have siliconized both plates as
> long as I have been sequencing w/o ever seeing this problem, I started to
> think about why the gel should creep upwards against gravity. 
> Polyacrylamide is not ionic, so it can't be due to the electric field. 
> The only hand-waving I can come up with is due to some sort of bulk ionic
> flow of the buffer due to the electric field, with causes the gel to
> counteract by moving in the opposite direction.
> 
> 	Anyone have any better ideas?
> 
> T.B. Shin
> tshin at husc8.harvard.edu
>  
> 

We have had the same problem, periodically. Expanding gels seem to occur
more often in summer months when the ambient temperature in the lab can
be more than 30 C. The standard solution has been to leave out the de-
gassing (under reduced pressure) step: pour the gel directly after 
filtering and the addition of NH4 persulfate and TEMED. Both observations
are counter-intuitive (you would think a cold gel would expand MORE on
heating, and that dissolved gasses would expand) but empiricism wins.

Steve

*********************************************************************
Steven M. Carr
Dept. of Biology
Memorial University of Newfoundland
St. John's NF A1B 3X9
CANADA

(709) 737-4776 office / -4713 lab / -4000 FAX
scarr at kean.ucs.mun.ca
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