Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 35)

DCode Dye Solution
Reagent
AmountFinal Concentration
Bromophenol blue
0.05 g 0.5%
Xylene cyanol0.05 g 0.5%
1x TAE buffer10.0 ml 1x
Store at room temperature.
2x Gel Loading Dye
Reagent
AmountFinal Concentration
2% Bromophenol blue
0.25 ml 0.05%
2% Xylene cyanol 0.25 ml 0.05%
100% Glycerol7.0 ml 70%
dH
2
O 2.5 ml
Total volume 10.0 ml
Store at room temperature.
1x TAE Running Buffer
Reagent
Amount
50x TAE buffer 140 ml
dH
2
O 6,860 ml
Total volume 7,000 ml
Gel Volumes
The table below provides the required volume per gel size and spacer thickness.
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. The PCR products should be evaluated for purity by agarose
gel electrophoresis before being loaded onto a denaturing acrylamide gel.
2. For a constant denaturing gel, load about 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.
3. Add an equal volume of 2x gel loading dye to the sample.
4. 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 het-
eroduplexes 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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