Salt Gradient Sequencing gels

jmv at canctr.mc.duke.edu jmv at canctr.mc.duke.edu
Mon Jun 28 10:11:47 EST 1993


In article <93173.105338IO00865 at MAINE.MAINE.EDU>, 
IO00865 at MAINE.MAINE.EDU (Ethan Strauss) writes:
>[...] Specifically I'm wondering
>when to add the NaOAC if I am doing 2 loadings.
>The procedure I am using says to use 1XTBE in the bottom chamber, 0.5X TBE
>in the upper chamber and add 3M NaOAC to the lower chamber about 1 hour
>after loading.

  Too much trouble. Seq. gels will run OK using just TBE gradient, but adding
NaOAc will help long or long-run lanes keep straight...so, add it to the 
bottom buffer before starting the gel & don't change it for either loading.

>I've had terrible problems with my sequencing gels leaking from the bottom
>while pouring. I seal the bottom with brown 3M mailing tape. [...]

  Tape should work; type shouldn't matter. Clamping at the edges could help.
Pouring a small gel plug at the bottom before pouring the rest of the gel
should help. 
  However, to maximize efficiency, use a bottom spacer: IBI
makes dovetailed plastic ones for its apparati (clamp, pour gel, remove
bottom spacer before running the gel & fill the space with bottom buffer);
or, you can make your own from paper (eg, Whatman 3MM): cut into strips of
desired width & length, abut bottom & side spacers without overlap, clamp
or clip plates with spacers inside, moisten edges of paper with H2O to seal,
pour gel as usual. Do not remove bottom spacer before running (current goes
through wet paper). It's a great way to recycle cut-offs from 3MM paper we 
use for backing/drying gels.

  Good luck,

--DBLoeb
___Duke U Med Ctr/Neurology Div.                 



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