Bio-Rad CHEF Mapper® XA System - Manual (Page 50)

8.3 Sample Loading
Use one of the following methods to load the sample.
• Place DNA in a sample plug on a smooth clean surface and cut to size using a razor blade
or spatula. Samples should be less than 90% of the height of the wells. Place agarose plugs
onto the front walls of the sample wells using a spatula, and gently press them to the bottoms
of the wells. Press the plugs firmly against the front walls of the wells. Fill each sample
well with Low Melt Preparative Grade Agarose at an agarose concentration equal to that of
the gel, and allow the agarose to harden at room temperature for 10 to 15 minutes.
• Cut the sample plug into blocks and place on each tooth of the comb. Cast around the comb.
The plug will remain in place when the comb is removed.
• Add liquid samples to the sample wells with the gel positioned under the electrophoresis
buffer in the chamber. Turn the pump off when adding liquid samples to prevent samples from
washing out of the wells. Run the samples into the gel for approximately 5 to 10 minutes
before turning the pump back on.
8.4 DNA Size Standards
Run standards in each gel to verify the sizes of unknown samples and to verify the elec-
trophoresis conditions. Figure 8.2 shows three of Bio-Rad’s standards for pulsed field
electrophoresis. These come as blocks of 0.75% low-melt agarose. Recommended run
conditions are given in the figure legend.
Fig. 8.2. Pulsed field standards, Lambda ladder (170-3635). Switch time was ramped from 50 seconds
to 90 second over the 22 hour run time. The gel was 1.0% agarose in 0.5x TBE. Electrophoresis was at
6 V/cm at 14 °C.
Schizosaccharomyces pombe ( 170-3633) Strain 972 h-. Switch time was 30 minutes
for 72 hours. The gel was 0.6% Chromosomal Grade Agarose in 0.5x TAE. Electrophoresis was at
1.5 V/cm at 14 °C.
Saccharomyces cerevisiae (170-3605) Strain YNN295. Switch time was ramped from
60 seconds to 120 seconds over the 24 hour run time. The gel was 1.0% agarose in 0.5x TBE. Electro-
phoresis was at 6 V/cm at 14 °C.
8.5 Electrophoresis
Remove the end plates from both ends of the casting stand, and slide the gel on the black
platform into the black frame on the surface of the electrophoresis chamber under the buffer.
Place the gel in the chamber so that the platform is positioned inside the frame. Check the
buffer level to insure that the gel is covered by about 2 mm of buffer. Adjust the buffer flow
by using the flow adjustment knob on the Variable Speed Pump. Enter the run parameters
using either Manual with algorithm, Bar Code, or Memory Program Storage (see Section 4 for
complete instructions).
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Lambda ladder S. pombe S. cerevisiae