Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 26)

5.4 Blotting Megabase DNAs †
Southern Blot Transfer
Pulsed field electrophoresis is a powerful technique for physical mapping of genes in
various organisms. 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 frag-
ments separated by CHEF are transferred onto membranes and detected by Southern hybridiza-
tion analysis. The procedures described for Southern transfer of DNA from standard agarose
gels onto membranes are applicable to large DNA fragments separated by CHEF, with the
addition of the gel pretreatment step given 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 agi-
tation. 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. Photograph the gel using very short exposures (<1 second) to minimize expo-
sure 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 0.4 N NaOH, 1.5 M NaCl for 15 minutes. Transfer the DNA onto
Zeta-Probe ® GT nylon membrane (catalog number 162-0196) using 2 liters of 0.4 N
NaOH, 1.5 M 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.
6. Carefully remove the paper towel and blotting papers. Remove the membrane together
with the gel, turn over the membrane and gel, lay them gel side up, and mark the location
of the wells and the orientation marker on the top of the gel. The position of the wells
can be accurately marked on the membrane by using a fine point permanent marker pen,
cutting through the bottoms of the wells.
7. Neutralize the membrane in 0.5 M Tris, pH 7.0 (neutralization buffer) for 5 minutes,
followed by rinsing briefly in 2x SSC. Transferred DNA can be visualized on the mem-
brane by placing the damp blot on a transilluminator.
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