was denatured and immobilized as described in appendix a except that schleicher amp schuell ba85 nitrocellulose filter was used cut in 9 mm diameter circles and non specific binding sites were blocked as described earlier under sample preparation about 0 3 pmol phage u03bb dna was fixed onto the nitrocellulose filter paper during this sample preparation process the 65 mer was hybridized to the u03bb dna by incubating the dna paper with 150 u03bcl 2xssc containing 4 pmol 65 mer for 3 hours at 57 u00b0 c and the 65 mer solution was removed 2xssc is 0 3m nacl 0 03m na citrate ph 7 0 the 15 mer was hybridized to the u03bb dna by incubating the dna paper with 150 u03bcl 2xssc containing 4 pmol of the labeled 15 mer for 16 hours at 36 u00b0 c a substantial portion of the 15 mer not perfectly bound i e not bound at all 15 sites in step 2 was removed by washing the dna paper in 320 ml 2xssc for 10 minutes at 23 u00b0 c and then washing the filter again in 2 u03bcl of the buffer 66 mm tris hcl at ph 7 5 6 6 mm mgcl 2 and 10m dithiothreitol the two probes were ligated by incubating the dna paper in 150 u03bcl of the buffer used in step 2a plus 0 4 mm atp and 2 u03bcl t 4 dna ligase for 2 hours at 23 u00b0 c the stringency was increased to remove nearly all of the labeled probe that was not ligated in step 4 this was accomplished by washing the dna paper by passing 200.