Creepy sequencing gels

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Thu Nov 25 12:34:00 EST 1993


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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
>  
> 
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HI netters
    I would like to add my $.02 worth Here!  I have noticed that the degree
of expansion of an acrylamide gel correlates with the number of times you wash
the wells!  That is to say the loss of Urea from the gel, by washing
the wells or Well, seems to give rise to this effect.  I do not wash my wells
until just before I load and only once and have never had this problem again.
For prerunning with a sharks tooth comb, pull the comb, set up gel, wash the 
outside of the 
plates, then prerun without washing the wells or inserting the comb, then wash
 the
wells, insert the comb and load!  For double loads only wash the wells that 
are
to be loaded first then when reloading wash the second set of wells. 
This should be the last you will see of extruding gels!

Dan Gietz
______________________________________
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)789-3458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
"Trying to do the Manitoba Thing"
_______________________________________



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