Bio-Rad CHEF Mapper® XA System - Manual (Page 58)

harder to control and is extremely sensitive to temperature. Exposure to short-wave 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 exactly.
1. Stain the gel with 1.0 µg/ml ethidium bromide (EtBr) for exactly 30 minutes with constant
agitation. Use a fresh dilution of 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 0.4 N NaOH, 1.5 M NaCI for 15 minutes. Transfer the DNA onto Zeta-
Probe ® GT nylon membrane (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:
• Corning Pyrex glass dish (28 x 18 x 4 cm)
• 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)
• Glass plate (16 x 20 cm)
• Two sheets of blotting paper as a wick (18 x 30 cm, S&S, GB002)
• Agarose gel (top side down)
• Zeta-Probe GT membrane cut to the size of the gel and prewetted with distilled water
• Two sheets of blotting paper (18 x 15 cm; S&S, GB002)
• 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 with the 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
with 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
membrane by placing the damp blot on a transilluminator.
8. Dry the membrane by blotting onto 3MM or other adsorbent paper and proceed with
hybridization. UV crosslinking of the DNA to the membrane is not recommended with
this alkaline transfer method.
Discussion
• The procedure is based on gels approximately 6 mm thick. If thicker gels are used, the
staining period may be prolonged to allow diffusion of EtBr into the middle of the gels.
DNA that is not stained with EtBr will not be nicked by the UV light and thus will not be
transferred from the gel.
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