Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 21)

Before beginning the electrophoresis run, check the current output displayed on the
CHEF-DR III power module to insure that the correct buffer concentration is used. The
following values are for 2 liters of buffer at 14 °C circulating through the electrophoresis cell.
Buffer Voltage Current
Concentration Gradient Range
0.5x TBE (at 14 ˚C) 2 V/cm 30–45 mA
0.5x TBE (at 14 ˚C) 3 V/cm 50–65 mA
0.5x TBE (at 14 ˚C) 6 V/cm 115–135 mA
0.5x TBE (at 14 ˚C) 9 V/cm 190–210 mA
1.0x TAE (at 14 ˚C) 2 V/cm 75–90 mA
1.0x TAE (at 14 ˚C) 3 V/cm 115–130 mA
1.0x TAE (at 14 ˚C) 6 V/cm 260–275 mA
1.0x TAE (at 14 ˚C) 9 V/cm 380–410 mA
If the current output is significantly different from the values listed above, drain the buffer, and
add new buffer. Premixed 10x TBE is available from Bio-Rad (catalog number 161-0733).
Concentrations of Buffers
Different final concentrations of electrophoresis buffer have been employed in pulsed
field electrophoresis. Recommended final buffer concentrations are:
0.5x TBE Buffer: 45 mM Tris 10x TBE Buffer: 108 g Tris base
45 mM borate (per liter) 55 g boric acid
1.0 mM EDTA 40 ml 0.5M EDTA,
pH 8.3 pH 8.0
1.0x TAE Buffer: 40 mM Tris 50x TAE Buffer: 242 g Tris base
40 mM acetate (per liter) 57.1 ml glacial acetic
2.0 mM EDTA acid 100 ml 0.5M
pH 8.0 EDTA, pH 8.0
4.3 Loading the Samples
Use one of the following methods to load the sample.
1. 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 10–15 minutes.
2. 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.
3. 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 minutes
before turning the pump back on.
4.4 DNA Size Standards
Bio-Rad recommends running standards in each gel to allow the sizes of unknown samples
to be determined 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.
18