Bio-Rad Mini-PROTEAN® Tetra Handcast Systems - Manual (Page 7)

reservoir chamber. Note: For this application, the heavy solution will be poured in the
mixing chamber labeled "light", and the light solution will be poured in the mixing chamber
labeled "heavy".
a. Arrange the tubing so that there is as short a distance as possible between the stopcock
opening and the needle at the end of the tubing. (Alternatively, you can cut the end of
the tubing at an angle.) The end of the needle or tubing will lie against the glass plate
while the acrylamide is being poured into the sandwich.
b. Place a 1" stirbar in the mixing chamber (with the valve stem closed.) The mixing
chamber is labeled "light".
c. Add the initiators to the light solution, swirl it 8 to 10 times, and pour the mixture into the
reservoir chamber (which is labeled "heavy"). This is the start of the 10 minutes. Leave
the valve stem closed.
d. Add the initiators to the heavy solution, swirl it 8 to 10 times, and pour it into the mixing
chamber (which is labeled "light"). 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.
For a concave gradient, insert the exponential piston into the mixing chamber to 1 cm above
the level of the acrylamide, tighten the screw top handle to hold the piston in place, and then
start the stir plate.
e. Quickly open the valve stem and stopcock. The acrylamide will flow through the tubing
and needle to the gel sandwich. Do not allow air bubbles to enter the gel. Overlay the
acrylamide with water or water-saturated isobutanol. For a continuous buffer system,
insert a comb. (The comb should be inserted at an angle to prevent the formation of
bubbles.)
f. Remove the tubing and disassemble the gradient former. Immediately flush the system
with water to prevent polymerization of residual acrylamide within the gradient former.
3.4.2 Pour a Linear or Convex Gradient Gel from the Bottom
In this case, the light solution goes into the mixing chamber (labeled "light") and the
heavy solution goes into the reservoir (labeled "heavy"). The light solution enters the gel from
the bottom and travels to the top, and the heavy solution follows, filling in the bottom of the
gel.
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.
a. Arrange the tubing so that there is as short a distance as possible between the stopcock
opening and the needle at the end of the tubing. Insert the needle into the gel sandwich
through the gasket on the bottom of the casting stand.
b. Place a 1" stirbar in the mixing chamber (with the valve stem closed.) The mixing
chamber is labeled "light".
c. Add the initiators to the light solution, swirl it 8 to 10 times, and pour the mixture into the
mixing chamber (which is labeled "light"). This is the start of the 10 minutes. Leave the
valve stem closed.
For a convex gradient, insert the exponential piston into the mixing chamber to 1 cm
above the level of the acrylamide, tighten the screw top handle to hold the piston in place.
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