Bio-Rad CHEF-DR II System - Manual (Page 24)

Buffer Voltage Current
Concentration Gradient Range
0.5x TBE (at 14 ˚C) 2 V/cm 30-40 mA
0.5x TBE (at 14 ˚C) 3 V/cm 50-60 mA
0.5x TBE (at 14 ˚C) 6 V/cm 110-120 mA
1.0x TAE (at 14 ˚C) 2 V/cm 80-90 mA
1.0x TAE (at 14 ˚C) 3 V/cm 120-130 mA
1.0x TAE (at 14 ˚C) 6 V/cm 260-270 mA
If the current output is significantly different from the values listed above, the buffer
should be drained, and new buffer should be added. 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. Bio-Rad’s 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 acid
2.0 mM EDTA 100 ml 0.5M EDTA
pH 8.0 pH 8.0
4.3 Loading the Samples
One of the following methods should be used to load the sample.
1. DNA in a sample plug should be placed 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 (catalog number
162-0017) at an agarose concentration equal to that of the gel, and allow the agarose to
harden at room temperature for 10 to 15 minutes.
2. Alternatively, the sample plug can be cut into blocks and placed on each tooth of the
comb. Cast around the comb.The plug will remain in place when the comb is removed.
3. Liquid samples can be added to the sample wells with the gel positioned under the elec-
trophoresis buffer in the cell. 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.
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