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UV Nicking of DNA in Agarose Gels for Enhanced Transfer and Detection of Megabase Size DNAs


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 sportation to the GS Gene Linker chamber, place the tray inside the GS Gene Linker chamber, and close the door.

3. Irradiate the gel with 60 mJoules of energy (nic program). The gel can now be photographed, but further exposure to UV light must be minimized (<10 seconds). The gel can be destained prior to photography if desired.

4. Soak the gel in 0.4 N NaOH, 1.5 M NaCl for 15 minutes. Transfer the DNA for at least 16 hours to Zeta- Probe GT membrane according to standard procedures, using 0.4 N NaOH, 1.5 M NaCl as the transfer solvent.

This protocol is for use with the GS Gene Linker UV chamber, with gels approximately 6 mm thick. If thicker gels are used, the staining period should be prolonged. DNA that is not stained with EtBr will not be nicked by UV irradiation, and will not transfer efficiently.


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