Hoefer SE900 - Manual (Page 19)

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Gradient Gels
Linear gradient gels can be cast using an optional accessory, the
Hoefer SG500 Gradient Maker. The SG500 mixes low percentage
and high percentage gel solutions that are pumped into the port at the
bottom of the multiple gel caster using a peristaltic pump.
Pouring a Linear Gradient Gel
1 Assemble the multiple gel caster as described on page 11, with the
following two exceptions:
• Do not insert the triangular rubber gasket at the bottom of
the gel caster.
• Remove the cap from the bottom inlet port.
2 Attach one end of laboratory grade tubing to the SG500 outlet port.
Insert the tubing through a peristaltic pump, and attach the other end
to the inlet port of the SE915 multiple gel caster faceplate.
3 Calculate the volume of monomer solution needed. Divide the total
volume in half and prepare this volume of both the higher- and lower-
percentage acrylamide solutions.
4 Optional: Adjust the higher-percentage acrylamide solution to 15%
(w/v) sucrose or up to 25% (v/v) glycerol to improve layering.
5 Pour the higher percentage, or heavy, acrylamide solution into the
SG500 chamber farthest from the outlet. Open the stopcock just long
enough to displace air between the chambers and then close.
6 Pour the lower percentage, or lighter, acrylamide solution into the
mixing chamber, the chamber with the SG500 outlet.
7 Place a stir bar into the mixing 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.
8 Turn on the peristaltic pump to a low setting, and open the stopcock
between the two SG500 chambers.
9 The gel solution should slowly layer in the triangular region at the
bottom of the multiple gel caster, and fill the cassettes evenly from the
bottom to the top.
10 Once almost all solution has exited the gradient maker pause the
pump temporarily, and fill the gradient maker with ~200 mls of the
displacement solution. Restart the pump and pump the solution
through the tubing, forcing the acrylamide solutions up into the gel
cassettes until the desired height is reached. Stop the pump.
11 Overlay each gel with 1 ml of water-saturated n-butanol or diluted
gel buffer to get the best top gel surface. Slowly deliver the overlay
solution on one side, minimizing mixing, and allow the overlay to flow
across the top surface unaided.
12 Allow the gels to polymerize for a minimum of one hour. After polym-
erization, pour off the overlay and rinse the gel surface several times
with distilled water.
13 Prepare the stacking gel monomer solution. Pour the stacking gel on
top of the resolving gel. Allow a minimum of one hour for the stacking
gel to polymerize.
Note: It is a good idea to practice with
water and the peristaltic pump to empirically
determine the volumes and the flow rates to
cast good gradient gels.
Note: The displacement solution is required.
The gradient will not form correctly without
displacing the acrylamide up inside the
cassettes. It also helps keep acrylamide from
polymerizing in the tubing and in the bottom
of the caster, simplifying clean up. The
displacement solution should not enter the
bottom of the cassettes.
Note: Avoid getting air bubbles into the
pump tubing, Air bubbles will interfere with
proper gradient formation.