Bio-Rad PROTEAN® Plus Hinged Spacer Plates and Combs - Manual (Page 8)

Section 5
Pouring Multiple Gradient Gels
in a Gel Casting Chamber
Note: Gel casting chambers are designed for bottom filling, therefore concave exponen-
tial gradient gels can not be found using a gel casting chamber.
1. The total gel volume required depends on several factors, including gel thickness, num-
ber of gels, gel length, and amount of space-filling. To make a precise determination of
the volume required for a particular application, set up the casting chamber with the glass
plates, spacers, and combs (for continuous gradients) to form the required number of gel
sandwiches and measure the volume of deionized distilled water required to fill it to the
desired level. Then disassemble the chamber and rinse and dry the parts.
2. Calculate the chamber volumes,. For a linear gradient, each chamber's volume is one-
half the total gel volume required. For a convex exponential gradient, the light solution
in the mixing chamber will be one-fourth the total gel volume and heavy solution in the
reservoir chamber will be equal to the total gel volume.
3. Reassemble the multi-gel casting chamber as described in step 2. Be sure the glass plates
and spacers are clean and dry.
4. Arrange the tubing in the peristaltic pump so there is as short a distance as possible
between the stopcock opening and the pump, and between the pump and the inlet to the
gel casting chamber. Attach a 3 cm piece of tubing to the gel casting chamber inlet with
a pinch clamp to close it off. Attach one end of a Luer taper coupling to this 3 cm tubing
and attach the tubing from the pump to the other end of the Luer taper coupling. Cut the
tubing as necessary.
5. For a linear gradient, set up the gradient former as described in Section 2.2, steps 4-9,
except pour the light solution into the mixing chamber and the heavy solution into the
reservoir chamber. For a convex exponential gradient, set up the gradient former as
described in Section 3.2, steps 3-7.
6. Run the pump so that the acrylamide flows through the tubing into the gel casting chamber,
until the acrylamide reaches the desired level in the gel sandwiches. Stop before any air
enters the gel casting chamber. For bottom filling of linear gradient, the tubing should con-
tain a negligible amount of acrylamide following gradient formation. For bottom filling of
a convex exponential gradient, one-fourth the volume will remain in the mixing chamber.
7. Overlay each gel sandwich with an equal volume of water. For a continuous gel, straight-
en the pre-positioned combs and insert them fully to form the wells.
8. Following gel casting, pinch off the tubing with the pinch clamp, remove the tubing from
the Luer taper coupling, and transfer it to a waste receptacle. Pump out any remaining
acrylamide from the chambers and pour rinse water into both the mixing and reservoir
chamber. Turn the peristaltic pump on to its maximum velocity to flush out the system.
Water should flow from the gradient former through the peristaltic pump and down all the
tubing to keep it from being plugged with any polymerized residual acylamide.
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