Bio-Rad PROTEAN® Plus Hinged Spacer Plates and Combs - Manual (Page 5)

If a peristaltic pump is not used, the level of the gradient former stopcock must be above
the top of the gel sandwich by a distance sufficient to create a hydrostatic head large
enough to pour the entire volume of the gel within 10 minutes from the time the initiators
are added to the light solution. All the acrylamide should be in the gel sandwich before
polymerization begins, so that polymerization will be uniform throughout the gel.
Rather then using a needle on the end of the tubing, you may cut the end of the tubing at
an angle before taping it to the sandwich. Face the tapered opening toward the glass plate.
4. Place a 1" stir bar (Model 385) or 2" stir bar (Model 395) in the mixing chamber (with
valve stem closed).
5. Degas the heavy and light solutions.
6. Add the initiators to the light solution, swirl it 8 to 10 times, and pour it into the reservoir
chamber. (This is the start of the 10 minutes.) Leave the valve stem closed.
7. Add the initiators to the heavy solution, swirl it 8 to 10 times, and pour it into the mixing
chamber.
8. Start the stirring motor and adjust the speed so that you get good mixing and still the bot-
tom level of the vortex matches the acrylamide level in the reservoir chamber.
9. Quickly open the valve stem and stopcock, and turn on the peristaltic pump.
Note: The peristaltic pump must be able to pump the whole gradient within 10 minutes
from the time you add the initiators to the first solution. This is generally in the range of
5 to 10 ml/min for the Model 385 and 70 to 100 ml/min for the Model 395.
10. Run the pump until all the solution is pushed into the sandwich.
If a peristaltic pump is not used, the acrylamide will flow down the tubing to the gel sand-
wich by gravitational force when you open the stopcock.
Note: If tubing is left dangling below the top of the sandwich, you will have to lift it up
and drain it into the top of the gel sandwich to get the entire volume of acrylamide into
the sandwich. The tubing volume should not be a problem because the tubing will retain
a negligible amount of acrylamide following gradient formation.
11. Overlay the acrylamide with water or water saturated isobutanol. For a continuous buffer
system, insert a comb.
Note: Immediately after the gradient is cast, the system must be flushed with water to pre-
vent polymerization of residual acrylamide within the gradient former or pumping system.
12. Immediately remove the needle (or tubing) from the top of the sandwich and transfer to
a waste receptacle. Pour rinse water into both the mixing and reservoir chambers and turn
the peristaltic pump on to its maximum speed. The water should immediately flow from
the gradient former, through all connectors and tubing of the pumping system, and out the
needle (or tubing) to a waste receptacle.
2.3 Pouring the Gel from the Bottom
In this case, the light solution goes in the mixing chamber, and the heavy solution goes
in the reservoir. The light solution enters the gel from the bottom and travels to the top, and
the heavy solution follows, filling the bottom of the gel.
1. Set up the gel sandwich, gradient former, tubing, and peristaltic pump as described in
Section 2.2, steps 1, 2, 4, and 5. For a continuous gradient, insert the comb at an angle.
3