on a 20 u00d7 13 5 cm 0 7 w v agarose gel stained with ethidium bromide 1 u03bcg ml and photographed the dna was denatured for two hours at 37 u00b0 in 0 5n naoh 1 5m nacl neutralized for an additional two hours at 37 u00b0 in 0 5m tris hcl ph 8 1 5m nacl and transferred overnight onto schleicher and schuell nitrocellulose filters in 10x sspe 1x sspe is 180 mm nacl 10 mm sodium phosphate ph 6 8 1 mm edta all from sigma chemical co a sponge was used instead of a paper wick the filters were washed briefly in 10x sspe air dried baked for 2 3 hours at 80 u00b0 in a vacuum oven and soaked for one hour at 50 u00b0 in 5x sspe denhardt 39 s was added to a final concentration of 5x 1x denhardt 39 s is 0 02 w v bovine serum albumin 0 02 ficoll 0 02 polyvinyl pyrrolidone all from sigma chemical co and the filters were soaked an additional hour the blots were prehybridized overnight at 37 u00b0 in 25 ml of a mixture containing 50 v v formamide 5x sspe 5x denhardt 39 s 0 1 w v sds sodium dodecyl sulfate and 100 u03bcg ml each of carrier salmon testes st dna and yeast trna prior to addition the st dna and trna were denatured by boiling for 10 minutes probes were radiolabelled by nick translation maniatis p 109 to a specific activity of 10 7 10 8 dpm u03bcg between 10 7 and 3 u00d7 10 8 dpm of the appropriate nick translated probe was added to the prehybridization mixture which was then incubated for 48 hours at 42 u00b0 the filters were washed twice for 15 min.