Bio-Rad CHEF-DR II System - Manual (Page 26)

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, it is recommended that 4-5 feet of the Tygon tubing be coiled
into a bucket of water. Recirculation of the buffer is required. It is recommended that the
buffer be changed 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. S. cere-
visiae chromosomes should be electrophoresed at 3.8-4.5 V/cm. Gel strength and buffer con-
centration 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, voltage must be decreased 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.
4.7 Removing and Staining the Gel
Before removing the gel make sure the run is completed (The unit will display End). To
stain the gel after a run, remove the frame from the electrophoresis cell, then remove the gel
(on the platform) from the cell. Slide the gel off the platform into a 0.5 µg/ml ethidium bro-
mide solution in water and let the gel stain for 20-30 minutes. (Caution: Ethidium bromide
is a mutagen. Always wear gloves while handling gels or solutions containing the dye.) Destain
the gel in distilled water for 1-3 hours. The DNA can be visualized by placing the gel on a UV
transilluminator (254-360 nm). Remove the buffer from the electrophoresis cell by unclamp-
ing the drain tube and allowing the buffer to drain into a 2 liter container with the pump turned
off. Discard used buffer and reclamp the drain tube.
Note: Leaving electrophoresis buffer in the cell with the lid on, when not in use, may
lead to warpage of the lid. Leave the lid ajar without buffer in the cell when not in use to
minimize potential warpage.
Section 5
Applications
5.1 Strategies for Electrophoretic Separations
There are several parameters that must be considered before performing an electrophoretic
separation of very high molecular weight DNA. The separations of large DNA molecules in
agarose gels are affected by agarose concentration, buffer concentration, buffer temperature,
initial and final switch times, voltage, total electrophoresis run time, and field angle.
The agarose concentration affects the size range of DNA molecules separated, and the
sharpness, or tightness, of the bands. Agarose concentrations of 1.0% are useful in separating
DNA molecules up to 3 mb in size. Agarose concentrations in the range of 1.2-1.5% are typ-
ically used for improved band tightness, however run times will increase proportionately. Gel
concentrations below 1.0% (0.5-0.9%) are useful in separations of extremely high molecular
weight DNA, greater than 3 mb, though the bands are a bit more diffuse.
23