Restriction digests of the clone give the following sizes (kb): EcoRI--10.0; HindIII--10.0; XbaI--9.2, 1.0; BglI--2.8, 2.35 (doublet), 1.55, 0.75, 0.28; PstI--4.0, 3.7, 2.5. Plasmid contains the following restriction sites (approximate kb from nt 1): BamHI--4.42, 4.77; BglI--1.96, 4.60, 5.32, 5.56, 7.88, 9.42; EcoRI--4.40; HindIII--4.45; PstI--0.65, 4.44, 8.00; PvuI--0.97, 4.57, 8.13; SmaI--4.41; XbaI--3.45, 4.42. 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 pDO17: Acc65I AflII ApaI AscI AvrII Ecl136II KpnI NotI PacI PmeI PmlI SacI SfiI SnaBI SpeI SrfI Sse8387I SwaI XbaI. 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'. |
