Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 23)

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 during a run, push PAUSE/START RUN on the CHEF-DR III system. Remove
the gel (on the platform) from the cell, slide it off the platform into a 0.5 µg/ml ethidium
bromide 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. Visualize the DNA by placing the gel
on a UV transilluminator (254–360 nm). Remove the buffer from the gel box by attaching a
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 closed, when not in use, may
lead to warpage of the lid. Leave the lid slightly opened 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.
Agarose Concentration
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
typically 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 molec-
ular weight DNA, greater than 3 mb, though the bands are a bit more diffuse.
There are several agarose types that allow easy handling of low concentration gels. These
agaroses, in concentrations of 0.5–0.8%, can be used to decrease the run time on separation
of large DNA (> 2 mb). An example of this type of agarose is Bio-Rad’s Chromosomal Grade
Agarose (catalog number 162-0135).
Buffer Concentration and Temperature
In pulsed field electrophoresis, DNA mobility is sensitive to changes in buffer tempera-
ture. As the buffer temperature increases, the mobility of the DNA increases, but band sharp-
ness and resolution decrease. Chill the buffer to 14 ˚C to maintain band sharpness and to
dissipate heat generated during prolonged runs. Also, buffer recirculation is required to pre-
vent temperature gradients from occurring. High voltage runs (300 V) exceeding 1 day require
buffer changes after each 48 hour period, to prevent buffer degradation. Standard Tris-borate
or TBE, at a concentration of 0.5x, is the most commonly used buffer in pulsed field elec-
trophoresis. Tris-acetate buffer, or TAE, at a concentration of 1.0x, can be used in place of
TBE. Other buffer concentrations are in the range of 0.25–1.0x. In Figure 5.1 two different
gels, one using 0.5x TBE and the other using 1.0x TAE, were run to show the difference in
mobility of DNA in the two buffers.
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