Hoefer SE245 - Manual (Page 7)

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p5
Pour the gel
1
Prepare the required amount of monomer solution.
Deaerate and add the initiator and catalyst just prior
to pouring the gel. Use a pipet to deliver the solution
into one corner of the plate, taking care not to intro-
duce any air pockets. See below for the appropriate
solution level according to the application.
No stacking gel
Fill solution to just below the top of the notched
plate. If air pockets form, remove with a pipet or
syringe. Introduce a comb (at a slight angle) into each
sandwich, taking care not to trap air bubbles under
the teeth.
Stacking gel
Fill solution to 3 cm below the top of the rectangular
glass plate. This height allows 1 cm of stacking gel
below the wells. Pour the gel and apply an overlay.
After the gel is polymerized, prepare the stacking gel
as described below.
2-D electrophoresis
Fill solution to about 1.5 cm below the top of the
rectangular glass plate and apply an overlay. This
height allows 4–5 mm of space for the IPG strip and
an agarose seal.
2
Overlay each gel with a thin layer of water-saturated
n-butanol, water, or diluted gel buffer to minimize
exposure of the top surface of the gel solution to
oxygen. Deliver the overlay solution slowly from a
glass syringe fitted with a 22-gauge needle. Apply the
solution near the spacer and allow it to flow across
the surface unaided.
3
Allow the gel to polymerize for a minimum of
one hour.
Caution! Acrylamide is a
neurotoxin. Always wear gloves
and observe all laboratory safety
procedures.