Bio-Rad CHEF Mapper® XA System - Manual (Page 51)

Note: In prior models, do not insert additional gel stops at the upper comers of the gel. This
will affect the uniformity of the electric field and result in distortion of the outer lanes.
8.6 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, coil 4–5 feet of the Tygon tubing into a bucket of water.
Buffer recirculation is required. Change the buffer 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. Saccharomyces cerevisiae should be
electrophoresed at 3.8–4.5 V/cm. Gel strength and buffer concentration 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 Saccharomyces cerevisiae DNA are the same as
those given in Section 9.5, 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, or some DNA may not enter the gel. In some cases, DNA
bands may be more diffuse at room temperature than when resolved at 14 °C.
8.7 Removing and Staining the Gel
Before removing the gel, make sure the run is completed. To stain the gel during a run,
push PAUSE/CONT on the CHEF Mapper panel, and wait until the high voltage indicator
light goes off. Remove the gel from the gel chamber, place it into a 0.5 pg/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), catalog numbers 170-3737, 170-3738, and 170-3745. Remove
the buffer from the gel box by unclamping the drain tube and allowing the buffer to drain
into a 2.2 liter container with the pump turned off. Discard used buffer, and reclamp the drain
tube. Leave the lid of the gel chamber open on one end when not in use.
Note: In prior models, leaving electrophoresis buffer in the gel chamber with the lid
closed, when not in use, can warp the lid.
Section 9
Applications
9.1 Strategies for Electrophoretic Separations
Several parameters 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, switch
times, voltage, pulse angle, and total electrophoresis run time
Agarose Concentration
The agarose concentration determines the size range of DNA molecules separated and
the sharpness, or tightness, of the bands. Agarose concentrations of 1% are useful in separat-
ing DNA molecules up to 3 mb in size. Agarose concentrations in the range of 1.2–1.5% are
typically used for improved band tightness; however, run times will increase proportionate-
ly. Gel concentrations below 1% (0.5–0.9%) are useful in separations of extremely high
molecular weight DNA, greater than 3 mb, although the bands are more diffuse.
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