Bio-Rad CHEF-DR II System - Manual (Page 27)

There are several agarose types that allow easy handling of low concentration gels. These
agaroses, in the concentration range of 0.5-0.8%, can be used to decrease the run time on sep-
aration of large DNA (> 2 mb). An example of this type of agarose is Bio-Rad’s Chromosomal
Grade Agarose (162-0135).
Buffer Concentration and Temperature
In pulsed field electrophoresis, the mobility of the DNA is sensitive to changes in buffer
temperature. As the buffer temperature increases, the mobility of the DNA increases, but the
band sharpness and resolution decrease. It is recommended that the buffer be chilled to
14 ˚C to maintain band sharpness and to dissipate heat generated during prolonged runs. Also,
buffer recirculation is required to prevent temperature gradients from occurring. High voltage
runs (200 V) exceeding 1 day require buffer changes after each 48 hour period, to prevent
any possible buffer degradation.Standard Tris-borate or TBE, at a concentration of 0.5x, is the
most commonly used buffer in pulsed field electrophoresis. Tris-acetate buffer, or TAE, at a
concentration of 1.0x, can be used in place of TBE. Other buffer concentrations used 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.
0.5x TBE 1.0x TAE
Fig. 5.1. Two gels, one in 0.5x TBE and the other in 1.0x TAE, were run to show the difference in mobil-
ity 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 will be
optimal for DNA molecules with reorientation times comparable to the switch time. So, as the
DNA size increases, the switch time needs to increase to resolve the molecules. Under some
conditions, larger molecules sometimes run ahead of smaller ones.
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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
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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