A procedure for repeated usage of 33P-labeled DNA probes in hybridization experiments.
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 microliters/cm2), 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 degrees 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).
Lichtenstein
A V
AV
Laboratory of Tumor Biochemistry, Institute of Carcinogenesis, All-Union Cancer Research Center, Moscow, Russia.
Moiseev
V L
VL
D001345:Autoradiography / Q000379:methods; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015247:Deoxyribonuclease HindIII; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D008214:Lymphocytes / Q000502:physiology; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D015183:Restriction Mapping
D015342:DNA Probes; D004279:DNA, Viral; D006360:Heat-Shock Proteins; D010761:Phosphorus Radioisotopes; D004247:DNA; D015247:Deoxyribonuclease HindIII