Gel Volumes; Sample Preparation - BIO RAD DCODE Manual

DCode Dye Solution
Reagent
Bromophenol blue
Xylene cyanol
1x TAE buffer
Store at room temperature.
2x Gel Loading Dye
Reagent
2% Bromophenol blue
2% Xylene cyanol
100% Glycerol
dH O
2
Total volume
Store at room temperature.
1x TAE Running Buffer
Reagent
50x TAE buffer
dH O
2
Total volume

Gel Volumes

The table below provides the required volume per gel size and spacer thickness.
Spacer Thickness
0.75 mm
1.00 mm
1.50 mm

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 (usu-
ally 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 heterodu-
plexes 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.
Amount Final Concentration
0.05 g
0.05 g
10.0 ml
Amount Final Concentration
0.25 ml
0.25 ml
7.0 ml
2.5 ml
10.0 ml
16 x 16 cm Gel
25 ml
30 ml
45 ml
30
0.5%
0.5%
1x
0.05%
0.05%
70%
Amount
140 ml
6,860 ml
7,000 ml
16 x 10 cm Gel
15 ml
20 ml
26 ml