Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 19)

Bacterial:
(4.5 x 10 6 bp)(660 g/mole)
= 4.933 x 10 -15 g DNA/cell (A)
6.02 x 10 23 bp/mole
(4.933 x 10 -15 g DNA/cell)(5 x 10 8 cell/ml)
=
(B)
(2.467 x 10 -6 g/DNA/ml)(1 x 10 6 µg/ml)
=
2.5 µg DNA/ml
(2.5 µg DNA/ml)(2.5 genome equivalents)
≅
6.25 µg DNA/ml (C)
(6.25 µg DNA/ml)
≅
(0.625 µg DNA/plug)
≅
0.3 µg DNA/lane (D)
10 plugs/ml 2 lanes/plug
Yeast:
(1.5 x 10 7 bp)(660 g/mole)
= 1.644 x 10 -14 g DNA/cell (A)
6.02 x 10 23 bp/mole
(1.644 x 10 -14 DNA/cell)(6 x 10 8 cell/ml)
=
(B)
(9.864 x 10 -6 g/DNA/ml)(1 x 10 6 µg/ml)
=
9.864 µg DNA/ml
(9.864 µg DNA/ml)(2 genome equivalents)
≅
(20 µg DNA/ml) (C)
(20 µg DNA/ml)
≅
(2 µg DNA/plug)
≅
1.0 µg DNA/lane (D)
10 plugs/ml 2 lanes/plug
Section 4
Gel Electrophoresis
4.1 Casting the Gel
Casting the gel requires the use of the following components: casting stand with removable
end plates, the casting platform, a comb and comb holder, and the frame which positions the gel
and platform in the electrophoresis cell. The casting stand provided with the CHEF-DR III
system is 14 cm (5.5") wide x 13 cm (5") long. Optional stands are 21 cm (8.5") wide x
14 cm (5.5") long, and 14 cm (5.5") wide x 21 cm (8.5") long. The gel should be cast on a
level surface. Bio-Rad’s Leveling Table (catalog number 170-4046) is useful for this purpose.
For detailed instructions, refer to the Casting Stand manual.
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, with the horizontal slot
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 in 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.
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