Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 20)

Volume Volume
per Adjustment
Spacer Size Gel Size Syringe Setting
0.75 mm 7.5 x 10 cm 5 ml 3.5
16 x 10 cm 8 ml 6.5
16 x 16 cm 11 ml 9.5
1.00 mm 7.5 x 10 cm 6 ml 4.5
16 x 10 cm 11 ml 9.5
16 x 16 cm 16 ml 14.5
1.50 mm 7.5 x 10 cm 8 ml 6.5
16 x 10 cm 15 ml 13.5
16 x 16 cm 24 ml 22.5
Spacer Thickness 16 x 16 cm Gel 16 x 10 cm Gel
0.75 mm 25 ml 15 ml
1.00 mm 30 ml 20 ml
1.50 mm 45 ml 26 ml
Sample Preparation
1. It is important to optimize the PCR reaction to minimize unwanted products which may
interfere with gel analysis. PCR products should be evaluated for purity by agarose gel
electrophoresis before being loaded onto a denaturing acrylamide gel.
2. For a perpendicular denaturing gel, load about 1–3 µg of amplified DNA per well
(usually 50% of a 100 µl PCR volume from a 100 ng DNA template). Both wild-type and
mutant samples are mixed together and run on the same gel.
3. For a parallel denaturing gel, load 180–300 ng of amplified DNA per well (usually 5–10%
of a 100 µl PCR volume from a 100 ng DNA template). A wild-type control should be run
on every gel.
4. Add an equal volume of 2x gel loading dye to the sample.
5. Heteroduplexes can be generated during PCR by amplifying the mutant and wild-type samples
in the same tube. If the samples are amplified in separate tubes, then heteroduplexes can be
formed by mixing an equal amount of mutant and wild-type samples in one tube. Heat the tube
at 95 °C for 5 minutes, then place at 65°C for 1 hour, and let slowly cool to room temperature.
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