Help with PAGE

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Thu Feb 6 19:59:46 EST 2003


Hi

Sorry for asking this, but why does your professor have you pour gels ?
Unless you need highly specialized mixtures, pre-cast gels are available
and, compared to the amount of time one spends pouring them, affordable.

http://www.kpl.com/support/immun/Protocols/PR_1.htm
http://researchlink.labvelocity.com/protocols/index.jhtml?id=31
have 'average' protocols - take a look perhaps you might find some
differences with what you're doing.

Now to the problems at hand:

>3.  Despite my settings for the power supply, when I set it for constant
voltage for my Electrophoresis step, it switches to constant >amperes, and
vice versa for the Transfer step.  Thus, the volts used in the
Electrophoresis end up far less than my setting for >electrophoresis, and
amperes are far less than my setting for the transfer.

First of all as you probably realize, you cannot run the gel with voltage
and current constant. The reason is simple - as you run the gel, the
resistance changes so one of the two parameters has to float. Ideally,
voltage should be kept constant, however if you do so you might encounter a
problem with heat dissipation. If you keep current constant then your heat
output won't change - but your voltage will. Many advanced power supplies
have AUTOMATIC CROSSOVER between constant voltage and current modes, with
limits either user-selectable or preset. Check your supply - this is what
likely causes switches in modes.

>1.  Although everyone says my gel recipes are perfect, I'm getting uneven
travel of my sample through different lanes, as indicated >by the dye in the
sample buffer, although it does appear even within a single lane.  I have
the power supply set to 200 V, 60 mA >for 1 hour.  My stacking gel is 5% and
my resolving gel is 7.5%.

Symptoms would indicate unevenly poured gels, bubbles, OR bad/uneven
electrodes of the electrophoresis chamber. Are the wiggles more or less
always the same from gel to gel ? Is the chamber designed poorly so that
e.g. bubbles can get trapped under the lower lip of the gel thus occluding
some of the exposed conductive surface ? How long do you give the gel to
settle ? Do you let the gels sit in a cold box for a while before running
them ?

>2.  I run my transfers overnight in a cold room, with the settings at 90 mA
and 30 V, per the BIO-RAD instructions for the >instrument.  When done, the
buffers are still cold, so I know it hasn't been cooked, but I'm having
difficulty separating the gel from >the membrane.  I have yet to get it off
in one piece, and without damaging the membrane.

Not sure what the problem is, this does not seem to happen with precast gels
and I have not used self-cast gels since grad school, however the symptoms
resemble 'incomplete curing' syndrome when the gel is not completely
polymerized and it keeps curing as it is being transfered. I would also
check the difference between the last buffer in which your gel is soaked
before transfer, and the transfer buffer itself. Also, check the pH of your
running and transfer buffer - it normally is about 8.3.  I usually run
transfers at about 200 mA starting current, and it usually takes about 90
minutes for full transfer from 12% gel, however your instructions would vary
depending on the particular protocol.

Good luck,

A.G.E.





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