Restriction digests of the clone give the following sizes (kb): EcoRV-8.2, 5.0; HindIII--13.5; BamHI--13.5; NheI--13.5; EcoRI--6.5, 2.5, 1.3. The pAL5000 ori permits replication in Mycobacterium smegmatis and M. bovis BCG. The vector should be selected using ampicillin and kanamycin in E. coli, and kanamycin in Mycobacterium spp. 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. A cosmid shuttle vector useful for genomic DNA library construction. The ScaI site between the cos sites can be used to linearize the plasmid. After packaging, 10.3 kb of vector is retained, permitting an insert range of 30 - 42 kb. Sequences hybridizing to pBR322, including ampR, are removed. The order of the major features in this plasmid is: cos - pMB1 ori - ampR/ScaI - cos - trpA terminator - XbaI - HindIII - fd terminator - EcoRV - pAL5000 ori - BamHI - rrnC terminator - EcoRV - lambda ori - NheI - kanR. |
