Bio-Rad Mini-PROTEAN 3 Multi-Casting Chamber - Manual (Page 5)

3.2 Casting Gradient Gels from the Bottom
The inlet port on the Mini-PROTEAN 3 multi-casting chamber is used for casting linear
and convex acrylamide gradient gels. Refer to the gradient maker instructions for preparation
of gradient solutions and proper operating techniques. The Model 485 Gradient Former
(catalog number 165-4120) is recommended for use with the Mini-PROTEAN 3 multi-casting
chamber.
An inexperienced user should practice all steps ahead of time so that the procedure is
completed quickly.
1. Place the gradient former on a magnetic stir plate and add a magnetic stir bar to the mix-
ing chamber labeled "light". Attach the luer fitting to the stopcock valve on the inlet port.
Run a piece of Tygon
® tubing (1/8" ID Tygon tubing works well) from the gradient for-
mer to the luer fitting on the multi-casting chamber.
2. Determine the volume of monomer needed (see Appendix A).
3. Combine all reagents except the initiators, and degas the solution for 15 minutes.
4. Immediately prior to pouring, add TEMED and APS to both solutions, mix gently, and
pour the appropriate monomer solutions into the gradient chambers. (Consult the Model
485 Gradient Former instruction manual for complete instructions.) The light solution
(the one with the lower acrylamide concentration) should be placed in the mixing cham-
ber labeled "light", and the heavy solution in the reservoir chamber labeled "heavy".
5. Turn on the stirring bar in the mixing chamber, open the tubing clamp of the gradient
maker and the stopcock valve of the casting chamber, and pour the gels.
Note: If gravity flow isn't fast enough, use a peristaltic pump to pump the entire set of
gradients within 10 minutes. If it is not possible to complete the operation in 10 minutes from
the time initiators are added, then it might be necessary to reduce the amount of initiators
(use ½ the amount of TEMED) to slow polymerization. The gradient should be poured as
quickly as possible, without mixing the gradient solution in the casting chamber.
Section 4
Chamber Disassembly and Storage of Gels
Note: Wear rubber gloves while performing the following procedure to prevent accidental
exposure to unpolymerized acrylamide, which is a neurotoxin.
1. Allow gel solution to polymerize for at least 1 hour.
2. Unscrew the clamp screws and remove the sealing plate carefully.
3. Remove the gels one at a time from the stack. Separate the gel sandwiches from the acrylic
blocks and plates used as space-fillers.
4. Rinse off the tops of all the gels thoroughly with distilled water. Trim off excess
acrylamide around the glass with a razor blade. Wash off any pieces of excess acrylamide
present with distilled water.
5. Store the gels upright in a tightly sealed container or zip-lock bag. Add a few milliliters
of 1X gel buffer (identical to the buffer in the gel) to the bottom of the container and to
the tops of the gels to prevent them from drying out. Store tightly sealed at 4 °C. Note: If
a stacking gel is required, consult Appendix B.
6. Clean the entire casting chamber thoroughly with distilled water. Residual acrylamide in
the stopcock valve and inlet port can be removed using a paper clip or a syringe needle.
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