Restriction digests of the clone give the following sizes (kb): BglI--7.8, 2.5, 1.6, 0.78; EcoRI--12.5; PstI--9.0, 2.5, 1.1. Plasmid contains the following restriction sites (approximate kb from nt 1): BamHI--6.98; BglI--6.61, 7.54, 7.77, 10.9, 11.63; EcoRI--3.89; HindIII--2.31; PstI--0.65, 1.78, 5.62, 10.22; PvuI--10.34; SalI--7.26; SmaI--0.99, 4.61; XhoI--0.89. A series of overlapping deletion segments can be generated by partial digestion with a restriction enzyme not found in the vector, followed by complete digestion with lambda terminase. Resulting fragments are blunted and circularized by ligation, and transformants are selected for either ampicillin or kanamycin resistance. The following restriction sites do not cut pDO184: AatII AflII ApaI AscI AvrII Bpu1102I Bsp120I BsrGI Bsu36I HpaI MunI NotI NsiI PacI PaeR7I PmeI PmlI Ppu10I SacII SfiI SnaBI SpeI SrfI StuI SwaI. IMPORTANT: To prevent amplification of a rearranged and/or deleted cosmid, we recommend streaking on LB + amp plates at 30C and picking small colonies for liquid culture. Cosmid vector containing two compatible origins of replication, for generating bidirectional deletions of large inserts to facilitate sequencing. AmpR transformants can be sequenced in the clockwise direction using the cosL primer 5'-TCATAAATAGCGAAAACC-3'. KanR transformants can be sequenced in the counterclockwise direction using the cosR primer 5'-ACTTTACGGGTCCTTTCCG-3'. |
