Bio-Rad CHEF-DR II System - Manual (Page 22)

Yeast:
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(B)
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Section 4
Gel Electrophoresis
4.1 Casting the Gel
Casting the gel consists of using the following components: a casting stand with remov-
able end plates; a platform on which the gel is cast, a comb and comb holder, and a frame
which positions the gel and platform in the electrophoresis cell. The casting stand provided
with the CHEF-DR II system is 14 cm (5.5”) wide x 13 cm (5”) long. An optional wide/long
combination casting stand, 21 cm (8.5”) x 14 cm (5.5”), is are available for greater through-
put or increased seapration.
The gel should be cast 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 man-
ual 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 towards 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 towards the platform until the
edge of the platform is inserted into the slot. Press down on the end gate, and gently tight-
en the screws. The slots force the platform against the bottom of the casting stand.
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 loosening the screw (counterclockwise), then tightening when the comb is properly
positioned. A thin plastic ruler makes a good height gauge.
19
(1.5 x 10 7 bp)(660 g/mole)
= 1.644 x 10 -14 g DNA/cell
6.02 x 10 23 bp/mole
(1.644 x 10 -14 g DNA/cell)(6 x 10 8 cells/ml) =
(9.876 x 10 -6 g DNA/ml)(1x10 6 µg/g) = 9.876 µg DNA/ml
(9.876 µg DNA/ml)(2 genome equivalents)
≅
(20 µg DNA/ml)
20 µg DNA/ml ≅ 2 µg DNA/plug ≅
1.0 µg DNA/lane
10 plugs/ml 2 lanes/plug