Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 44)

Gel Volumes
The table below provides the required volume for the gel size and spacer
thickness.
Spacer Thickness 16 x 16 cm gel
0.75 mm 25 ml
1.00 mm 30 ml
1.50 mm 45 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 temporal temperature gradient 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.
3. Add an equal volume of 2x gel loading dye to the sample.
Pre-heating the Running Buffer
1. Fill the electrophoresis tank with 7 L of 1.25x TAE buffer.
Note: It is recommended that the running buffer not be reused. Reusing the running buffer
may affect the migration rate and band resolution.
2. Place the temperature control module on top of the electrophoresis tank. Attach the power
cord to the temperature control module, turn the power, pump, and heater on. The clear
loading lid should be on the temperature control module during preheating.
3. Set the temperature controller to the desired temperature. Set the ramp rate to 200°C/hr.
to allow the buffer to reach the desired temperature the quickest.
4. Preheat the buffer to the set temperature. It can take 1 to 1.5 hours for the system to
preheat the buffer to the set temperature. Heating the buffer in a microwave helps reduce
the preheating time.
Assembling the TTGE Gel Sandwich
For the temporal temperature gradient gel format, a 16 x 16 cm gel sandwich size is
recommended. To insure proper alignment, make sure all plates and spacers are clean and
dry before assembling. Use caution when assembling the glass plate sandwiches. Wear
gloves and eye protection at all times.
1. Assemble the gel sandwich on a clean surface. Lay the large rectangular plate down first, then
place the spacers of equal thickness along the short edges of the larger rectangular plate.
2. Place the short glass plate on top of the spacers so that it is flush with the bottom edge
of the long plate.
3. Loosen the black thumb screw of each sandwich clamp by turning it counterclockwise.
Place each clamp at the appropriate side of the gel sandwich with the locating arrows
facing up and toward the glass plates (Figure 4.24).
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