Bio-Rad Mini-PROTEAN® Tetra Handcast Systems - Manual (Page 4)

2
Section 2
Pouring Multiple Gradient Gels in the Multi-Casting
Chamber
Note: This procedure can be used to produce gels with either a linear or a convex
exponential gradient. Cast a minimum of 4 Mini-PROTEAN 3 gels using this procedure.
An inexperienced user should practice all steps ahead of time so that the procedure is
completed quickly. The best guarantee of reproducibility from gradient to gradient is
careful technique on the part of the operator.
Wear rubber gloves to prevent exposure to unpolymerized acrylamide, a neurotoxin.
2.1 Calculate the Volume of Acrylamide to Prepare
The first time gels of a certain thickness are cast, it is necessary to empirically determine
the required volume of acrylamide. If you are using the Mini-PROTEAN 3 multi-casting
chamber, assemble the stack of Mini-PROTEAN 3 sandwiches (as described in the
multi-casting chamber manual) and inject a measured volume of water through the stopcock.
You will prepare this volume (+5 ml) of acrylamide. (A minimum working volume of 40 ml
is required for the gradient former.) Disassemble the chamber and dry all components.
See Appendix A for additional information.
2.2 Calculate the Chamber Volumes
To create a linear gradient, the volume in each chamber is ½ the total gel volume required
(or 20 ml, whichever is greater.)
For a convex exponential gradient, the light solution in the mixing chamber will be ¼
the total gel volume (minimum 10 ml) and the heavy solution in the reservoir chamber will
be equal to the total gel volume (minimum 40 ml).
Combine all reagents except the initiators (usually APS and TEMED), and degas each
solution under vacuum for at least 15 minutes.
2.3 Reassemble the Multi-Casting Chamber
Refer to the Mini-PROTEAN 3 multi-casting chamber instruction manual. Be certain
that all of the parts are clean and dry.
2.4 Pour the Gels
Note: If the flow rate is kept constant, the gradients will be reproducible. Tubing size, the
volume of acrylamide in the chambers, and the rate of stirring must be kept constant. If the rate
of stirring changes, then the amount of acrylamide pulled into the front chamber from the
back chamber when the connection between the two chambers is opened will vary. This will
change the gradient.
2.4.1 To Pour Linear Gradient Gels
a. Arrange the tubing so that there is as short a distance as possible between the stopcock
opening and the inlet to the gel casting chamber.
Note: The level of the gradient former stopcock must be placed above the top of the gel
sandwich. This creates a hydrostatic head large enough to pour the gels within 10 minutes
from the time the initiators are added to the light solution. To create uniform gradients, all of
the acrylamide must be in the gel sandwich before polymerization begins.