Bio-Rad CHEF-DR II System - Manual (Page 29)

5.4 Blotting Megabase DNAs †
Southern Blot Transfer
Pulsed field electrophoresis has become a powerful technique for physical mapping of
genes in various organisms. In order to determine the chromosomal location of a gene in a
microorganism or the size of the restriction fragment containing a gene in mammalian systems,
large DNA fragments separated by CHEF are transferred onto membranes and detected by
Southern hybridization analysis. The procedures described for Southern transfer of DNA
from standard agarose gels onto membranes are applicable to large DNA fragments separat-
ed by CHEF, with the addition of the gel pretreatment step listed below.
Gel Pretreatment
Since DNA fragments larger than 20 kb cannot be transferred efficiently, DNA fragments
separated by pulsed field gels must be cleaved before transfer onto membranes. DNA can be
cleaved by using either acid (depurination) or UV irradiation. The depurination reaction is
harder to control and is extremely sensitive to temperature. Exposure to shortwave UV light
is a reliable method for nicking DNA in pulsed field gels before transfer.
Procedure
The following procedure was developed for use with the GS Gene Linker® UV chamber.
For optimal results, this protocol must be followed rigorously.
1. Stain the gel with 1.0 µg/ml ethidium bromide (EtBr) for exactly 30 minutes with constant
agitation. Use a fresh dilution of the EtBr stock for each gel. Do not destain the gel prior
to nicking.
2. Immediately UV irradiate the gel, using the GS Gene Linker chamber, with 60 mJoules
of energy. The gel should be photographed using very short exposures (<1 second) to
minimize exposure to UV radiation. The gel can also be destained if desired. Transfer
the nicked DNA to nylon membrane using alkali or neutral conditions (see discussion).
3. Soak the gel in 0.4N NaOH, 1.5M NaCl for 15 minutes. Transfer the DNA onto Zeta-
Probe ® GT nylon membrane (162-0196) using 2 liters of 0.4N NaOH, 1.5M NaCl as the
transfer solvent.
4. Set up the capillary transfer as follows, from bottom to top:
A. Corning Pyrex glass dish (28 x 18 x 4 cm).
B. A plexiglass or plastic box for support, about 3 cm high and small enough to fit in
the glass dish (e.g., Eppendorf yellow pipette tip rack).
C. Glass plate (16 x 20 cm).
D. Two sheets of blotting paper as a wick (18 x 30 cm; S&S, GB002).
E. Agarose gel (top side down).
F. Zeta-Probe GT membrane cut to the same size as the gel and prewetted with
distilled water.
G. Two sheets of blotting paper (18 x 15 cm; S&S, GB002).
H. A stack of paper towels 10 cm high.
5. Transfer the DNA 24-48 hours.
† Contributed by Dr. Eric Lai, University of North Carolina
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