Hoefer SE640 - Manual (Page 22)

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4
Pour the “light” solution into the reservoir chamber
(the chamber furthest from the inlet). Open the
stopcock long enough to displace air between the
chambers and then close. Pour the “heavy” solution
into the mixing chamber and place a stirring bar
into this chamber. Place the gradient maker onto a
magnetic stirrer and begin stirring at a rate that mixes
well but does not introduce bubbles into the solution.
5
Mix the gradient and pump the solution into the sandwich
While the solution is stirring, begin pumping from
the mixing chamber and open the stopcock to the
reservoir chamber. Raise the cannula as liquid enters
the sandwich, keeping the tip at the gel surface.
Prepare more gels as required.
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 at the side of
the sandwich and allow it to flow across the surface
unaided.
7
Allow the gels 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.