Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 22)

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Fig. 4.2. Lambda ladder was separated on a 1.0% Molecular Biology Certified Agarose (catalog num-
ber 162-0133) gel in 0.5x TBE, recirculated at 14 °C. The run time was 22 hours at 6 V/cm with a 50 to
90 second switch time ramp at an included angle of 120°.
Saccharomyces cerevisiae Strain YNN295.
Chromosomes were separated on a 1.0% Pulsed Field Certified Agarose (catalog number 162-0137)
gel in 0.5x TBE, recirculated at 14 °C. The run time was 24 hours at 6 V/cm with a 60 to 120 second
switch time ramp at an included angle of 120°.
Hansenula wingei Strain YB-4662-VIA. Chromosomes
were separated on a 0.8% Pulsed Field Certified Agarose gel in 1.0x TAE, recirculated at 14 °C. The run
time was 48 hours at 3 V/cm with a 500 second switch time at a included angle of 106°.
Schizosaccharomyces pombe Strain 972 h-. Chromosomes were separated on a 0.6% Chromosomal
Grade Agarose (catalog number 162-0135) gel in 1.0x TAE, recirculated at 14 °C. The run time was 72
hours at 1.5 V/cm with a 30 minute switch time at a included angle of 106°.
4.5 Electrophoresis
Remove both end gates by loosening the screws. Push the end gates off the edge of the
platform for removal, and slide the platform out of the casting stand. Place the gel and
platform assembly into the frame so that the bottom of the platform rests on the floor of the
cell. Do not remove the gel from the platform. Check the buffer level to insure that the gel is
covered by about 2 mm of buffer. Adjust the buffer flow, if necessary, by using the flow
adjustment knob on the Variable Speed Pump. Enter your run parameters (refer to Section 2
for complete operating instructions).
Prior to the first separation of experimental samples, we recommend an initial separation
of one or more of the four DNA size standards illustrated in Figure 4.2, using the conditions
described in the legend. Obtaining separations similar to those in Figure 4.2 will indicate that
the CHEF-DR III system is functioning properly.
4.6 Separations at Room Temperature
Electrophoresis may be conducted at room temperature, without a chiller, but the buffer
should not be allowed to exceed 30 °C. It is important to maintain the temperature at a steady
value. To facilitate heat transfer, coil 4-5 feet of the Tygon tubing into a bucket of water.
Recirculation of the buffer is required. Change the buffer every 24 hours.
Since heat generation is proportional to the square of the voltage, it is essential to lower
the field strength to 4.5 V/cm or less, depending on the size of DNA to be resolved.
Electrophorese S. cerevisiae chromosomes at 3.8-4.5 V/cm. Gel strength and buffer concen-
tration do not need to be changed, although switch times and run times may be increased
10 to 20% at the lower field strength. The conditions for resolution of S. cerevisiae chromo-
somes are the same as those given in Table 2, Section 5.3, except that the voltage should be
reduced to 4.5 V/cm when the temperature is 29 °C.
Alternatively, the ionic strength of the buffer may be decreased to 0.25x TBE. In this case,
decrease voltage even more than above or some DNA may not enter the gel. In some cases,
DNA bands may be slightly more diffuse at room temperature than when resolved at 14 °C.
Lambda Ladder S. cerevisiae H. wingei S. pombe