0 2 ml of 0 2 m naoh 1 sodium dodecyl sulfate sds was added and the mixture incubated for a further 5 min on ice 0 15 ml of 3m naac ph 4 8 was added and the mixture incubated on ice for 1 hr followed by centrifugation at 12 000 u00d7 g for 5 min plasmid dna present in the supernatant was precipitated by addition of 1 ml ethanol recovered by centrifugation and redissolved in 0 1 ml of 10 mm tris hcl ph 7 5 1 mm edta te buffer plasmid dna preparations were screened for the presence of the vaccinia virus promoter by digestion of 10 of each plasmid preparation with restriction endonucleases hindiii and ecori 5 units of each enzyme in medium salt restriction buffer for 1 hr at 37 u00b0 c dna fragments were separated by agarose gel electrophoresis and visualized as described above plasmid preparations containing the vaccinia virus promoter were analyzed further to determine the orientation of the vaccinia promoter with respect to plasmid sequences this was accomplished by digestion with restriction endonucleases hindiii and hincii or hinciii and ecori 5 units of each enzyme in medium salt restriction buffer at 37 u00b0 c for 1 hr followed by agarose gel electrophoresis a plasmid having with the vaccinia promoter reading toward the plasmid 39 s unique bamhi restriction site was called pgs15 this plasmid was purified in large amounts by the following procedure hereafter celled preparation of plasmid dna bacteria containing the required plasmid were seeded into a 400 ml solution of m 9 medium containing 50 u03bcg ml ampicillin 150 u03bcml proline 150 u03bcg ml leucine 0 8 u03bcg ml vitamin b 1 and grown until the optical density.