Bio-Rad Mini-PROTEAN® Tetra Handcast Systems - Manual (Page 6)

Section 3
Pouring One Large-Format Gradient Gel
Pouring a single Mini-PROTEAN 3 gradient gel is not recommended. The minimum
volume for the gradient former is 40 ml, and you should use nearly all of the solution in pouring
a gradient gel. These instructions are intended for casting large-format polyacrylamide gels.
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.
Note: If the rate of flow is kept constant, then the gradients will be reproducible. Tubing
size, the needle size and size of the hole in the needle, the volumes in the chambers, and the
rate of stirring of the stir plate 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.
3.1 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. Inject a measured volume of water into the gel sandwich.
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.
3.2 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).
For a concave exponential gradient, the heavy solution in the mixing chamber will be ¼
the total gel volume (minimum 10 ml) and the light 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.
3.3 Assemble the Glass Plate Sandwich
Set up the glass plate for casting (as indicated in the electrophoresis cell instruction
manual.)
3.4 Pour the Gel
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 gel 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.
3.4.1 Pour a Linear or Concave Gradient Gel from the Top
The heavy solution is poured into the mixing chamber since it enters the sandwich first,
flowing to the bottom. The light solution, which flows into the gel last, is poured into the
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