Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 52)

5.4 Sample Preparation
1. It is important to optimize the PCR reaction to minimize unwanted products which may
interfere with gel analysis. 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.
2. The PCR products should be evaluated for purity by agarose gel electrophoresis before
being loaded onto a heteroduplex gel.
3. About 180–500 ng of heteroduplex DNA (usually 5–15% of the total PCR volume) can
be loaded per well.
4. Add an equal amount of 2x gel loading dye to the samples.
5.5 Adding the Running Buffer
1. Remove and place the temperature control module on the DCode lid stand.
2. Add 2 or 7 L of running buffer to the electrophoresis tank. For CSGE, the upper buffer
concentration is different from the lower buffer concentration; therefore, the pump should
not be used.
Note: To improve heat dissipation during electrophoresis, 7 L of buffer can be used.
3. Place the temperature control module on the electrophoresis tank.
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