6 Denaturing Gradient Gel Electrophoresis
91
Casting a perpendicular denaturing gradient gel
5A. Clean glassplates thoroughly with detergent and water. Rinse with distilled water, then 100% ethanol, air dry.
6A. Assemble the glass plates and 0.75 mm spacers in the gelcastingstand
according to the manufacturer's instructions. Instead of inserting a
comb, insert a third spacer. The length of this spacer should be 5
mm less than the width of the gel, leaving space to insert the needle
for casting the gel. The set-up is shown in Figure 3. Add some silicone
grease at the closed end to prevent leakage; fix the spacer with two steel
clamps.
7 A. Turn the gel casting apparatus with the gel by approx. 89° so that the 5
mm open space is at the top end, to prevent trapping of air (see Figure
3).
8A. Prepare the two gradient solutions in separate 15 ml tubes (e. g. Falcon).
Fora 16 x 18 x 0.075 cm gel, 12 ml of each gradient solution should be
sufftcient.
Note: The gradient range depends on the melt map of the fragment to be
analyzed (see "Optimizing Conditions"). For perpendicular DGGE, it is adr
Spacer
I
I
-·
Silicone tube to
gmd; en'J
20 G need le
Gelcasting
stand
3rd spacer
as comb
.., Apply s iliSpacer
L1-cone
[
grease
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Fig. 3. Casting a perpendicular DGG