Bio-Rad Mini-PROTEAN 3 Multi-Casting Chamber - Manual (Page 4)

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Note: To insure a good seal, the entire stack should be made as flush as possible to the top
of the casting chamber, and not extend beyond it. If you overfill the chamber, monomer
solution may leak out during pouring, and glass plates in the stack may break.
8. Seat the gasket firmly in the notch in the sealing plate.
9. With the clamp screws loosened, slide the sealing plate under the clamps of the casting
chamber, being careful not to disturb the stack. The inlet port should match the groove at
the bottom of the chamber. Gradually tighten the screws in a random fashion until tight.
10. Stand the casting chamber up, and place it on a level surface. Do not tip the chamber
upside-down at this stage.
Section 3
Casting Gels
Single percentage gels can be prepared by introducing monomer from either the top or the
bottom of the chamber. Gradients must be introduced from the bottom.
The first time gels of a certain thickness are cast, it is necessary to empirically determine
the required volume of acrylamide. Assemble the stack as outlined in Section 2, and inject a
measured volume of water through the stopcock. Prepare this volume (+5 ml) of acrylamide.
Note: Wear rubber gloves while performing the following procedure to prevent accidental
exposure to unpolymerized acrylamide, which is a neurotoxin.
3.1 Casting from the Top (Non-Gradient Gels Only)
1. Attach the stopcock valve to the inlet port of the casting chamber. Make sure that the
valve is in the closed position.
2. Mark a level on the chamber (with tape or a pen) at the desired separation gel length,
measuring from the bottom.
3. Combine all reagents except the initiators (usually APS and TEMED), and degas the
solution under vacuum for at least 15 minutes.
4. After degassing, add initiators to the gel monomer solution, and introduce the monomer
into the gel sandwich closest to the sealing plate. A simple way to do this is to flow the
solution down the middle of the Spacer Plate of that sandwich using a pipet or a large
syringe. The groove on the bottom of the casting chamber will equilibrate the solution to
each of the gel sandwiches. Monitor the filling process by observing the level of
solution rising on the sandwich furthest from the sealing plate. Stop when you reach the
desired gel height.
5. Overlay the gels (if a stacker is required), or insert the comb immediately.
6. Allow enough time for complete polymerization of the separating gel before removing the
overlay solution (about 1 hour).
7. Prepare the stacking gel monomer solution as before and apply to the gels one at a time.
8. Insert a comb into each gel sandwich. To minimize bubble formation, insert the comb at
an angle.
Note: You may also cast single percentage gels from the bottom. Use the gradient former, but
just add the single percentage solution into both reservoir chambers.