Bio-Rad CHEF-DR II System - Manual (Page 25)

4.4 DNA Size Standards
Bio-Rad recommends running standards in each gel to allow the sizes of unknown sam-
ples to be determine and to verify the electrophoresis conditions. Figure 4.2 shows four Bio-
Rad standards for pulsed field electrophoresis. These come as blocks of 1.0% Low Melt
agarose. Recommended running conditions are given in the figure legend.
Lambda Ladder S. cerevisiae H. wingei S. pombe
Fig. 4.2. A. Lambda ladder (catalog number 170-3635) was separated on a 1.0% Molecular Biology
Certified Agarose (catalog number 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.
B. Saccharomyces cerevisiae Strain YNN295. (Catalog number 170-3605). Chromosomes were
separated on a 1.0% Pulsed Field Certified Agarose (catalog number 162-0137) gel in 0.5x TBE, recir-
culated at 14 °C. The run time was 24 hours at 6 V/cm with a 60 to 120 second switch time ramp.
C. Hansenula wingei Strain YB-4662-VIA. (Catalog number 170-3667). Chromosomes were separat-
ed on a 0.8% Molecular Biology Certified Agarose gel in 1.0x TAE, recirculated at
14 °C. The run time was 50 hours at 3 V/cm with a 250 to 900 second switch time ramp.
D. Schizosaccharomyces pombe Strain 972 h-. (Catalog number 170-3633). Chromosomes were
separated on a 0.6% Chromosomal Grade Agarose (catalog number 162-0135) gel in 1.0x TAE, recir-
culated at 14 °C. The run time was 72 hours at 2 V/cm with a 20 to 30 minute switch time ramp.
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 plat-
form 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 cov-
ered by about 2 mm of buffer. Adjust the buffer flow, if necessary, by using the flow adjust-
ment 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 above (Figure 4.2), using the run con-
ditions described in the legend. Obtaining separations similar to those in Figure 4.2 will indi-
cate that the CHEF-DR II system is functioning properly.
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