A procedure for repeated usage of 33P-labeled DNA probes in hybridization experiments

Abstract

We describe a technique for repeated use of 33P-labeled DNA probes in Southern hybridization experiments. A nick-translated 33P-labeled DNA probe in a volume of 0.5–1.0 ml of hybridization mixture (final concentration, 10–100 ng/ml) is used to wet a sheet of filter paper (approx 10 μl/cm 2), which covers a nylon membrane with DNA transferred by Southern blotting, and both are set between two washed X-ray films. The “sandwich” is placed in a plastic bag for hybridization for 16–24 h at 42°C. This very simple procedure using 33P-labeled DNA probes has a number of advantages over the standard method using 32P-labeled probes: (a) a significantly lower biohazard (body/arms exposure); (b) a very small volume of hybridization mixture in contact with a DNA-containing membrane and the higher probe concentrations attainable, causing some increase in sensitivity, and, finally, (c) repeated use of the probe-containing filter (over approx 3 days for unique sequences and up to 2 weeks for reiterated sequences) due to a relatively long 33P half-life (25.3 days).