Ultraviolet nicking of large DNA molecules from pulsed-field gels for southern transfer and hybridization.
Large DNA molecules separated in pulsed-field gels are not efficiently transferred from the gel for Southern hybridization. Various procedures for fragmenting the DNA prior to transfer are in use, but quantitative details that permit reproducible application have not been reported. We have determined the optimum level of energy for uv nicking of large DNA needed to promote efficient Southern transfer and detection by hybridization. To ensure consistent results we have used a uv oven equipped with a detector that measures only 200-400 nm wavelengths, and we report the total energy delivered. Using uv nicking and the transfer techniques described, we can obtain hybridization signals overnight with single-copy DNA probes on Southern blots of large DNA fragments separated by pulsed-field gel electrophoresis.
Lee
H
H
Department of Pharmacology, University of North Carolina, Chapel Hill 27599-7365.
Birren
B
B
Lai
E
E
D015139:Blotting, Southern / Q000379:methods; D015825:Chromosomes, Fungal; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000528:radiation effects*; D004271:DNA, Fungal / Q000235:genetics*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D009693:Nucleic Acid Hybridization; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014162:Transfection; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D004247:DNA