precipitated once with ethanol after removal of the ethidium bromide and resuspended in a minimal volume of te linear plasmids were purified from the less dense band after a second gradient was performed to ensure that this preparation did not contain contaminating sc plasmids contaminating linear duplexes were removed by ion exchange column chromatography according to the method described by garon and petersen 1986 gene anal techn 4 5 8 briefly linear dna was denatured in the presence of 0 2 m naoh for 10 min and then the ph was adjusted to 8 0 the salt concentration was adjusted to 0 5 m and the sample was immediately applied to a nags 37 mini column bethesda research laboratories gaithersburg md brl equilibrated with 0 5 m nacl linear plasmids were then eluted from the column with 0 8 m nacl ethanol precipitated and resuspended in a minimal volume of te recombinant sc plasmids from escherichia coli were extracted from 500 ml cultures and purified as previously described simpson et al 1987 infect immun 55 2448 2455 except dye buoyant density gradients were performed as above sc plasmid enriched dna 1 u03bcg from b burgdorferi strain sh 2 82 was digested with ecori and the resulting fragments were cloned in e coli strain dhs u03b1 brl using dephosphorylated puc13 brl ligation reactions were incubated at 16 u00b0 c for 12 hr with freshly prepared ligation buffer 50 mm tris ph 7 6 10 mm mgcl 20 mm dithiothreitol 1 mm atp and rubidium chloride induced competent cells were transformed as previously described maniatis et al 1982 molecular cloning a laboratory manual cold spring harbor laboratory cold spring harbor n y were done to identify.