Bio-Rad CHEF Mapper® XA System - Manual (Page 48)

Fig. 7.4. Effects of sample overloading on hybridization signals. Human DNA was prepared in
agarose simultaneously using two different cell concentrations. A sample block of identical thickness
from each was digested with Not I and separated as described in Figure 7.3 The amounts of DNA
loaded per lane were: Lane 1, 3 µg. Lane 2, 15 µg. A. Ethidium bromide stained gel. B. Autoradio-
gram after hybridization. Blotting and hybridization were as described in Figure 7.1.
Section 8
Gel Electrophoresis
8.1 Casting the Gel
To cast the gel use a casting stand with removable end plates, platform, a comb, and a comb
holder. The casting stand which comes with the CHEF Mapper system is 14 cm (5.5”) wide x
13 cm (5”) long. Casting stands 21 cm (8.5”) wide x 14 cm (5.5”) long, or 14 cm (5.5”) wide x
21 cm (8.5”) long are available.
Cast the gel on a level surface. Bio-Rad’s Leveling Table (catalog number 170-4046) is
recommended for this purpose. For more detailed instructions, refer to the manual provided
with the casting stand.
1. Slide the platform into the casting stand. There is no sidedness to the platform. Position
one end gate over the screws protruding from the casting stand, insuring that the hori-
zontal slot is facing the platform. Slide the edge of the platform into the slot. Press down
on the end gate, and gently tighten the screws.
2. Position the other end gate over the screws, and slide it toward the platform until the edge
of the platform is inserted into the slot. Press down on the end gate, and gently tighten the
screws. The slots force the platform against the bottom of the casting stand.
3. To attach the comb to the comb holder, place the comb over the two metal pins, and turn
the screw clockwise. This causes one pin to move toward 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. Prepare the desired concentration of Bio-Rad’s agarose in buffer.
Melt the agarose in a microwave oven or on a hot plate.
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A B
1 2 1 2