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Contributed by Donald R. VanDevanter, Tumor Institute, Swedish Hospital Medical Center, 409 Elkind Hall, 747 Summit Avenue, Seattle, WA 98104
Introduction
Analyses of uncharacterized DNA fragments using CHEF electrophoresis often
require preliminary studies to determine what size ranges DNA fragments
of interest fall within. It is not uncommon to run more than one gel in
order to fully characterize large DNA fragments from a single cell source1.
This is because DNA fragments under 200 kilobase pairs (kbp), between
200 kbp and 2 megabase pairs (mbp), and between 2 mbp and 6 mbp are optimally
separated by CHEF electrophoretic techniques using different switching
frequencies, electrophoretic field strengths, angles of orientation (if
using a Bio-Rad CHEF Mapper system), and/or agarose type and concentration2.
Data is presented illustrating that an initial estimate of sizes can be
obtained in a single gel by using a biphasic linear switch time ramping
procedure.
Results
A biphasic linear switch time ramping strategy3 with a fixed electric
field strength can be used with the CHEF-DR II system to resolve linear
DNA fragments ranging from 23 kbp to 5.7 mbp in size on a single gel (Figure
1). The gel shown in Figure 1A was 0.5% Chromosomal Grade Agarose made
in 0.5x TBE (45 mM Tris borate, 1.0 mM EDTA, pH 8.0), recirculated at
10 C. The applied electric field was held constant at 1.95 V/cm (65 V),
and switch times were ramped linearly from 30 seconds to 2 minutes over
33 hours, then from 2 minutes to 50 minutes over 55 hours. Under these
conditions, the chromosomes of Schizosaccharomyces pombe (closed circles,
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