Restriction digests of the clone give the following sizes (kb): EcoRI--6.8; HindIII--6.8; XbaI--5.8, 0.9. Plasmid contains the following restriction sites (approximate kb from nt 1): BamHI--3.08; BglI--3.26, 5.34; EcoRI--3.06; KpnI--3.07; HindIII--3.11; PstI--0.65, 5.47; PvuI--3.23, 5.59; SacI--3.07; SmaI--3.08; XbaI--2.11, 3.09. Resulting fragments are blunted and circularized by ligation, and transformants are selected for either ampicillin or kanamycin resistance. 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. A series of overlapping deletion segments can be generated by cutting at the Cos site with lambda terminase, then filling in ends with a alpha phosphorothioate nucleotides, cutting with a restriction enzyme that cuts on one side of the insert, and then digesting with Exo III for various intervals of time. 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'. pDO192 contains 12 unique restriction sites in the polylinker but no restiction sites for any 8 bp cutters. The order of the major features in the plasmid is: 3' lacZ - HindIII/MCS/EcoRI - 5' lacZ - ori 15 - kanR - cosR/L - ampR - oriP. |
