Bio-Rad Mini-PROTEAN® Tetra Handcast Systems - Manual (Page 5)

b. Add a 1" stirbar in the mixing chamber (with the valve stem closed.) The mixing
chamber is labeled "light". Place the gradient former on a magnetic stir plate.
c. Add the initiators to the light solution, swirl it 8 to 10 times, and pour the mixture into the
mixing chamber. This is the start of the 10 minutes.
d. Add the initiators to the heavy solution, swirl it 8 to 10 times, and pour it into the
reservoir chamber. Start the stir plate and adjust the speed so that you get good mixing;
the bottom level of the vortex should be nearly the same as the acrylamide level in the
reservoir chamber.
e. Quickly open the valve stem and stopcock. The acrylamide will flow down the tubing to
the gel sandwich. Do not allow air bubbles to enter the gel. Overlay the acrylamide with
water or water-saturated isobutanol. Note: each gel must have the same volume of
overlay solution. For a continuous buffer system, insert a comb at an angle (this reduces
the chance of bubble formation.)
f. Close the stopcock on the multi-casting chamber after the gels are cast. Remove the
tubing. Immediately flush the system with water to prevent polymerization of residual
acrylamide within the gradient former.
2.4.2 To Pour Convex Exponential 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.
b. Add a 1" stirbar in the mixing chamber (with the valve stem closed.) The mixing
chamber is labeled "light". Place the gradient former on a magnetic stir plate.
c. Add the initiators to the light solution, swirl 8 to 10 times, and pour into the mixing
chamber, leaving the valve stem closed. This is the start of the 10 minutes.
d. Immediately fix the volume with the exponential piston by inserting the piston into the
chamber to 1 cm above the level of the acrylamide and tightening the screw-top handle
to hold the piston in place.
e. Add the initiators to the heavy solution, swirl it 8 to 10 times, and pour it into the
reservoir chamber. Start the stir plate and adjust the speed so that you get good mixing;
the bottom level of the vortex should be nearly the same as the acrylamide level in the
reservoir chamber.
f. Quickly open the valve stem and stopcock. The acrylamide will flow down the tubing to
the gel sandwich. Do not allow air bubbles to enter the gel. Overlay the acrylamide with
water or water-saturated isobutanol. Note: each gel must have the same volume of
overlay solution. For a continuous buffer system, insert a comb at an angle (this reduces
the chance of bubble formation.)
g. Close the stopcock on the multi-casting chamber after the gradient is cast. Remove the
tubing. Immediately flush the system with water to prevent polymerization of residual
acrylamide within the gradient former.
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