Bio-Rad CHEF-DR II System - Manual (Page 28)

very large DNA (>3 mb) cannot be resolved on the gel and the field strength needs to 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 resolution has to be made.
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 field angle. As the migration rate of the DNA molecules decreases, the electrophoresis run
time needs to increase to adequately resolve the DNA molecules of interest.
5.2 Pulsed Field Conditions by DNA Size
In the table below are suggested run parameters that can be used for the various DNA
size ranges listed.
DNA DNA DNA DNA
1-100kb 0.1 - 2.0 mb 2 - 4 mb > 4 mb
% Agarose 1.0-1.2% 0.8-1.2 % 0.6-1 % 0.5-0.8 %
Buffer 0.5x TBE 0.5x TBE 1.0x TAE 1.0x TAE
Temperature 14° C 14° C 14° C 14° C
Voltage 6 V/cm 4.5-6 V/cm 2-3 V/cm 1.5-2.5 V/cm
Pulse Parameters 0.05-10 sec 10-200 sec 200-1800 sec 10-60 min
Run Times 2-15 hr 15-30 hr 24-72 hr 72-144 hr
5.3 Pulsed Field Conditions by Organism
In the table below are the run parameters for various types of DNA samples.
DNA Agarose Switch time Run Time
DNA size (kb) Conc. (seconds) (hours)Voltage Angle [Buffer]
Restriction 0.2-23 1.2% 0.01a 4 6 V/cm 120° 0.5x TBE
Fragments
5 kb Ladder 5-75 1.0% 1-6 b 11 6 V/cm 120° 0.5x TBE
Lambda Ladder 50-1,000 1.0% 50-90 c 22 6 V/cm 120° 0.5x TBE
Saccharomyces 200-2,200 1.0% 60-120 d 24 6 V/cm 120° 0.5x TBE
cerevisiae
Candida 1,000-4,000 0.8% 120 e 24 3.5 V/cm 106° 1.0x TAE
albicans 240 36
Schizo- 3,500-5,700 0.8% 1,800 f 72 2 V/cm 106° 1.0x TAE
saccharomyces
pombe
Dictostelium 3,600-9,000 0.8% 2,000-7,000 g 158 1.8 V/cm 120° 0.25x TBE
discodium 7,000-9,600 82 1.5 V/cm 120°
(a) 0.09 second single switch time for 3 hours.
(b) Ramped switch time from 1 to 6 seconds over 11 hours.
(c) Ramped switch time from 50 to 90 seconds over 22 hours.
(d) Ramped switch time from 60 to 120 seconds over 24 hours.
(e) 120 secondswitch time for 24 hours followed by 240 second switch time for 36 hours.
(f) 30 minute single switch time for 72 hours.
(g) Two blocks, with voltage change in the second block. Buffer temperature is 10 °C. Cox et al., Proc. Natl. Acad. Sci. USA, 87,
8247-8251 (1990).
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