Bio-Rad PROTEAN Plus Multi-Casting Chamber - Manual (Page 7)

5. After degassing, add initiators to the gel monomer solution, and introduce the solution into
the top of the gel sandwich closest to the sealing plate. Minimize bubble formation
during casting (try to flow the solution down the middle of the sandwich using a large
syringe, or direct flow down the side of the spacer with a pipette or graduated cylinder.)
The groove on the bottom of the multi-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.
6. Overlay each gel as quickly as possible with 1.0–2.0 ml of water or water-saturated
isobutanol. Optimally, the overlay solution should be applied to every gel simultaneously.
Note: each gel must have the same volume of overlay solution.
7. Allow enough time for complete polymerization of the separating gel before removing the
overlay solution (about 1 hour). While keeping the plates assembled in the chamber, rinse
the gels off thoroughly with water. After rinsing, measure the volume required to fill the
stacking gel area with water. Position the gel on its side and place a rectangular piece of
filter paper between the short and long plates. Dry the stacking gel area, inside the plates
next to the gel, by running the filter paper from top to bottom.
Caution: do not touch or disturb the gel with the filter paper.
8. Prepare the stacking gel monomer solution (see sample calculations below) and apply to
the gels one at a time.
Note: the final concentration of TEMED is 0.1%, the concentration of Tris-Cl stock is
0.5M pH 6.8, and the final concentration is 0.125M.
Sample Calculation: STACKING GEL
Objective: Cast stacking gels for 12 gels, 2.0mm thick, 25 x 20.5 cm.
Measure the volume required to fill the stacking gel area with water. The total
volume required to cast all 12 stacking gels is approximately 350 ml (this volume will
vary according to the height of the separating gel). Prepare 350 ml of solution. Use
the standard equation, C
1
V
1
= C
2
V
2
.
To prepare 350 ml (4%)
(4%) (350 ml) = (X ml) (30% acrylamide/Bis Stock) 47 ml
(0.125M Tris-Cl)(350 ml) = (X ml) (0.5M Tris-Cl pH 6.8 Stock) 87.5 ml
(350 ml) – (47 ml + 87.5 ml) = ml Water 215.5 ml
(0.05% APS) (350 ml) = (X ml) (10% APS Stock) 1.75 ml
(0.1% TEMED) (350 ml) = (X ml) (100% TEMED Stock) 350 µl
9. Insert a comb into each hinged spacer plate. 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.
3.2 Casting Gradient Gels
The inlet port at the bottom of the PROTEAN plus multi-casting chamber is used for
casting linear and convex acrylamide gradient gels using the Model 495 Gradient Former
(catalog number 165-4121). Refer to the gradient former instruction manual for additional
information and for proper operating techniques.
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