Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 20)

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3. To attach the desired comb to the comb holder, place the comb over the 2 metal pins, and
turn the screw clockwise. This causes one pin to move towards the screw, holding the comb
in place. Adjust the height of the comb to 2 mm above the surface of the platform by loos-
ening the screw (counterclockwise), then tightening when the comb is properly positioned.
A thin plastic ruler makes a good height gauge.
4. Place the comb holder with attached comb into one of the two positioning slots on each
side of the casting stand. Check that the bottom of the comb is at least 2 mm above the
surface of the platform. Pour approximately 100 ml of the desired agarose solution (<60 °C)
into the casting stand for a thickness of approximately 5–6 mm. Allow the gel to solidi-
fy for 30 minutes at room temperature.
5. Carefully remove the comb holder and comb; it is sometimes helpful to rock the holder
back and forth slightly during its removal. Sample plugs can be added to the wells with
the gel in the casting stand.
Fig. 4.1. The CHEF-DR III casting stand and comb holder.
4.2 Buffer Circulation and Temperature
Level the electrophoresis cell with the leveling feet at each corner by placing the casting plat-
form in the center of the cell, then placing the leveling bubble (provided) on the casting platform.
Putting the casting platform in the center of the cell will level the gel with respect to the elec-
trophoresis cell. Remove the casting platform after leveling. Position the frame in the electrophoresis
cell by placing the 2 plastic pins into the bottom set of holes (toward the front ports) in the floor of
the cell so that the frame is centered (center hole in each group of 3 holes). Pour 2.0–2.2 liters of
buffer (appropriate concentration of TBE or TAE) into the cell. Switch on the CHEF-DR III power,
then switch on the variable speed pump. Circulate at ~ 0.75 L/min (a setting of ~ 70 on the pump
regulator). Maintain the flow rate at the maximum setting that does not disturb the gel
Note: If the buffer circulation appears slower that normal (i.e. less than 500 ml / min at a set-
ting of 70 on the pump regulator, it is possible that the outlet ports on the electrophoresis cham-
ber are clogged with agarose. Reverse the recirculation flow to unclog the ports. Remove any
agarose debris from the chamber and restore the flow to original condition.
Allow the buffer to equilibrate to the desired temperature. We recommend 14 ˚C buffer
temperature in cell. The electrophoresis buffer can be chilled by the following methods:
1. Attach the Cooling Module (see Cooling Module manual for set-up and operation).
2. Coil pump tubing into a temperature-controlled water bath with the temperature set so
that the buffer temperature in the gel chamber is 14 ˚C.