Help with PAGE
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Wed Feb 12 05:57:54 EST 2003
Belinda Williams wrote:
> 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%.
60 mA is way too much for a minigel (6x8cm). Use 15 mA while the
tracking dye is in the stacking gel, than increase to 20. This will
reduce uneven heating, which is in part responsible for the uneven
running of the samples ("smilling": higher conductivity in the central
part of the gel due to higher temperature).
Good electrophoresis systems have cooling circuits, use those too.
> 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.
Low percentage gels (stacking gel!) can sometimes stick. Separate under
> 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.
During the run the conductivity of the gel changes, so either voltage
has to go up or current down. IMHO for electrophoresis you best use
constant current, for blotting constant voltage.
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