Bio-Rad PROTEAN Plus Multi-Casting Chamber - Manual (Page 6)

Section 3
Casting Gels
Single percentage gels (non-gradient) can be prepared by introducing monomer from
either the top or bottom of the multi-casting 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 monomer solution. Assemble the stack as outlined in Section 2.1, and
inject a measured volume of water through the stopcock. Prepare this volume (+ 10 ml) of
monomer solution (see sample calculations in Section 3.1 and 3.2). Disassemble the
multi-casting chamber and dry all components.
Note: Wear rubber gloves while performing the following procedure to prevent accidental
exposure to non-polymerized acrylamide, which is a neurotoxin.
3.1 Casting from the Top (Non-gradient Gels Only)
1. Attach a short length of tubing (8 cm of 1/8" ID Tygon ® tubing is included) to the inlet
port of the multi-casting chamber. Connect one of the two included stopcocks to the end
of the tubing. Make sure that the valve is in the closed position.
2. Mark a level on the multi-casting chamber (with tape or a pen) at the desired separation
gel length, measuring from the bottom.
3. Prepare the monomer solutions.
Note: For the larger format gels ( ≥ 20cm in length) it is necessary to decrease the TEMED
amount by 50% (for a final concentration of 0.025%). This will extend the polymerization time
to approximately 20 minutes, providing time to complete casting and for the monomer
solution to settle properly.
Sample Calculation: SEPARATION GEL
Objective: Cast 12 gels, 2.0mm thick, 25 x 20.5 cm, 12% acrylamide.
Place 12 clean hinged spacer plates and separation sheets in the multi-casting chamber
(as described in Section 2.1) and measure the volume required to fill the plates with
water. The volume required to cast the separating gel is approximately 1000 ml (this
volume will vary depending on the desired separating gel height).
Disassemble and dry all components. Prepare 1000 ml of solution. Use the standard
C
1
V
1
= C
2
V
2
equation.
To prepare 1000 ml (12%)
(12%) (1000 ml) = (X ml) (30% acrylamide/Bis Stock) 400 ml
(0.375M Tris-Cl)(1000 ml) = (X ml) (1.5M Tris-Cl pH 8.8 Stock) 250 ml
(1000 ml) – (400 ml + 250 ml) = ml Water 350 ml
(0.05% APS) (1000 ml) = (X ml) (10% APS Stock) 5 ml
(0.025% TEMED) (1000 ml) = (X ml) (100% TEMED Stock) 250 µl
4. Combine all reagents except the initiators (usually TEMED and APS), and degas the
solution under vacuum for at least 15 minutes. Degassing the solutions removes oxygen
(oxygen inhibits polymerization.)
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