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.
Temperature Controller
The temperature controller maintains the desired buffer temperature and controls the tem-
perature ramp rate in the DCode system (Figure 4.23). The actual and set buffer temperatures
are displayed in degrees Celsius. The set temperature and the temperature ramp rate (RR) can
be adjusted by using the raise and lower buttons. The °C/RR button is used to scroll between
the two parameters.
Fig. 4.23. The temperature controller displays the actual temperature, set temperature, and
temperature ramp rate.
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.
38
ACTUAL
HEATER
SET
°C
°C
RR
60.0
60.0
Summary of Contents for DCODE
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