Bio-Rad CHEF-DR II System - Manual (Page 23)

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 sur-
face 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 solidify 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 while
the gel remains in the casting stand.
Fig. 4.1. The CHEF-DR II casting stand, frame, and comb holder.
4.2 Buffer Circulation
Level the CHEF-DR II electrophoresis cell by adjusting the leveling screws at the corners.
Place the leveling bubble in the center of the base of the cell. Position the frame in the elec-
trophoresis cell by placing the 2 plastic pins into the bottom set of holes (towards the “T”
connector) in the floor of the cell so that the frame is centered (center hole in each group of
3 holes). Pour 2 liters of buffer (appropriate concentration of TBE or TAE) into the cell.
Switch on the CHEF-DR II 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 set-
ting that does not disturb the gel. 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 Model 1000 Mini Chiller (see Model 1000 Mini Chiller 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 electrophoresis cell is 14 ˚C.
Prior to beginning the electrophoresis run, the current output displayed on the CHEF-DR II
control module should be checked to insure that the correct buffer concentration is used. The
following values are for 2 liters of buffer at 14 °C circulating through the electrophoresis cell.
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