Hoefer SE400 - Manual (Page 22)

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5
Mix the gradient. While the solution is stirring, begin
pumping (5–10 ml/min) from the mixing chamber and
immediately open the stopcock to the reservoir cham-
ber. Raise the cannula as liquid enters the sandwich,
keeping the tip at the gel surface.
6
Overlay each gel with a thin layer of water-saturated
n-butanol, water, or diluted gel buffer to prevent gel
exposure to oxygen. Slowly deliver the overlay solution
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.
7
Allow the gel(s) to polymerize for a minimum of one
hour. After polymerization, pour off the overlay and
rinse the gel surface several times with distilled water.
8
Prepare the stacking gel monomer solution, pour the
stacking gel and introduce a comb (at a slight angle)
into the sandwich, taking care not to trap air under
the teeth. Allow a minimum of one hour for the gel
to polymerize.