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Introduction
The transfer efficiency of large (>100 kb) DNAs from agarose gels to membrane
is poor unless the DNAs are nicked prior to transfer. Although this has
traditionally been accomplished with HCl depurination, UV irradiation
(254 nm) provides a faster and more consistent alternative to HCl nicking.
The GS Gene Linker UV chamber contains preprogrammed routines for many
UV crosslinking and other controlled dosage applications, including UV
nicking of pulsed field gels prior to transfer to membrane. The chambers
unique sensor measures only the effective wavelengths of UV light, insuring
reproducible results every time.
Results
High molecular weight DNA from a human HeLa cell line was cleaved with
various restriction enzymes, resolved on the CHEF Mapper XA pulsed field
electrophoresis system using the Auto Algorithm mode (50-750 kb), and
irradiated with 60 mJoules of energy in the GS Gene Linker UV chamber
after staining with ethidium bromide. The DNA was alkaline transferred
to Zeta-Probe GT membrane, and probed with a human T cell receptor variable
region (single copy gene) probe. As illustrated in Figure 1, bands ranging
in size from 100 kb to greater than 700 kb were detected by the probe
in the DNA lanes.
UV Nicking Protocol
Note: This protocol must be followed rigorously to obtain optimal results.
1. Following electrophoresis, stain the gel with 1.0 g/ml ethidium bromide
(EtBr) for exactly 30 minutes with constant agitation. Use a fresh dilution
of EtBr for each gel. Do not destain the gel.
2. Place the gel on a glass tray for tran
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Source:
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