Cut-oocyte voltage-clamp: help needed;

davef at cc.usu.edu davef at cc.usu.edu
Fri Nov 10 12:26:40 EST 1995


I, too, wrestled with a cut-open oocyte (vaseline gap) clamp for several
months, often frustrated by inability to clamp quickly or adequately.

I think your problems may be due to inadequate clamping & isolation of the
guard chamber.  Leak between guard & upper chambers is tough to get rid of, and
does weird things.  Go *VERY LIGHTLY* with the vaseline.  Put it on, then wipe
it off with a finger so there is barely a fine film ... your seals should then
improve between inside & guard chamber.  Be careful your electrode (monitors
inside voltage) doesn't push the membrane away from the underside of the guard
chamber.  You can run fine platinum wires through your agar bridges, for
decreased resistance, if you want.  If your membrane doesn't hold up, you may
be leaving the saponin in too long.  Go ahead and start rinsing it out before
you notice the resistance to the inside (capacitance artifacts) change; 
sometimes the solution movement finishes the job, and if not, you can always go
back and add more ...it's a lot easier than starting over from the outset with
a new oocyte.

You might want to check out my TEV clamping page... The artifacts and some of
the problems with TEV are applicable to cut-open clamping too:

http://cc.usu.edu/~davef/tev/tev.html

Anyway, good luck.

-dave



                                                            




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