Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 24)

Fig. 5.1. Two gels, one in 0.5x TBE and the other in 1.0x TAE, were run to show the difference in
mobility of DNA in the two buffers.
S. cerevisiae was separated on a 1.0% Pulsed Field Certified
Agarose (catalog number 162-0137) gel with a 60 second switch time for 15 hours, followed by a 90
second switch time for 9 hours, at 6 V/cm. Notice the increased migration of the DNA molecules in the
TAE gel when compared with the TBE gel.
Switch Times
The migration rate of DNA molecules through an agarose gel is dependent on switch
time, voltage (field strength), field angle, and run time. In pulsed field electrophoresis, DNA
molecules are subjected to alternating electric fields imposed for a period called the switch
time. Each time the field is switched, the DNA molecules must change direction or reorient
in the gel matrix. Larger molecules take longer to reorient and therefore have less time to
move during each pulse, so they migrate slower than smaller molecules. Resolution is opti-
mal for DNA molecules with reorientation times comparable to the switch time. So as the
DNA size increases, increase the switch time to resolve the molecules. Under some conditions,
larger molecules may run ahead of smaller ones.
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Voltage (Field Strength)
DNA migration increases 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.
Field Angle
The CHEF-DR III system allows separations to be carried out with electric field vectors
oriented in any direction in the plane of the gel (90°–120°). With two field vectors, resolution
of DNA molecules up to 1 mb is independent of the angle between them (Birren, Lai, Clark,
Hood, Science, 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 5.2
shows the effect of the included angle on the separation of yeast chromosomes. Decreasing
the included angle will decrease the resolution of smaller DNAs by causing them to pile up
on each other. This same effect on small DNA can be seen with long switch times. It is rec-
ommended that the included angle be decreased (<120°) when separating large DNA
molecules greater than 2 mb.
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800
600
400
200
Size (kb)
2,400
2,200
2,000
1,800
1,600
1,400
1,200
0
0.0 0.1 0.2
Velocity (cm/hr)
0.3
1.0x TAE
0.5x TBE
0.4
1,000
0.5x TBE 1.0x TAE