Bio-Rad CHEF Mapper® XA System - Manual (Page 53)

Voltage (Field Strength)
DNA migration will increase with increases in voltage or field strength. However, greater
migration is accompanied by decreased band sharpness. In general, as the size of the DNA
molecules increases, the field strength should decrease. At high field strengths (6 V/cm) some
very large DNA (>3 mb) cannot be resolved on the gel and the field strength must be reduced.
Moreover, some large DNA molecules will not enter the gels at high field strengths. Therefore,
in selecting the field strength for an experiment, a compromise between run time and resolu-
tion has to be made.
Pulse Angle
The CHEF Mapper system allows separations to be carried out with electric field vectors
oriented in any direction in the plane of the gel (0–360°). With two field vectors, resolution
of DNA molecules up to l mb is independent of the angle between them, as long as the angle
is obtuse (Birren, Lai, Clark and Hood, Science, 241, 1203-1205, 1988). It has been shown that
decreasing the included angle from 120° to 94° increases the velocity of the DNA, with the
mobilities of large DNAs (>1 mb) affected to a greater degree by the change in angle than are
smaller DNAs (<1 mb). Figure 9.2 shows the effect of the included angle on the separation
of S. cerevisiae chromosomes. Decreasing the included angle will decrease the resolution of
smaller DNA by causing them to pile up on each other. This same effect on small DNA can
be seen with long switch times. Decrease the included angle (<120°) when separating DNA
molecules larger than 1 mb.
Fig. 9.2. Separation of S. cerevisiae using angles from 120° to 94°.
Electrophoresis Run Time
The electrophoresis run time is determined by the migration rates of the DNA molecules
under investigation. The migration rates, in turn, are affected by the switch time, field strength,
and pulse angle. As the migration rate of the DNA molecules decreases, the electrophoresis
run time must be increased to adequately resolve the DNA molecules of interest.
9.2 Ramping Factor
The CHEF Mapper system lets you program switch time ramps in a variety of mathematical
shapes. The manner in which switch times are ramped from initial to final values influences band
spacing. By appropriate choice of the ramping factor, DNA bands can be spread out or compressed
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120° 105° 100° 96° 94°