By Raymond L. Rodriguez, David T. Denhardt
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Extra info for Vectors. A Survey of Molecular Cloning Vectors and their Uses
Sample text
N. (1980) Cell 20, 529-542. W. (1977) Cell 10, 521-536. B. (1980) Plasmid 3, 99-108. , and Datta, N. (1967) Nature 214, 885-887. , and Oka, A. (1979) Eur. J. Biochem. 97, 435-443. R. (1981) Proc. Natl. Acad. Sei. USA 78, 14161420. , and Sain, B. (1984) Gene 30, 261-265. , and Philipson, L. (1983) Nucl. Acids Res. 11, 8019-8026. , Geuther, R. , Hoffmeier, C , and Gaspar, S. (1981) Mol. Gen. Genet. 184, 121-124. , and Rich, A. (1982) Cell 31, 309-318. , and Rich, A. (1983) Proc. Natl. Acad. Sei. USA 80, 1821-1825.
The origin of DNA replication is represented by ori. phage D N A is injected with the assistance of the host's ptsM gene product. Both lamB and ptsM are involved in host sugar transport and induced by growing cells in the presence of 0 . 2 % maltose (best in the absence of glucose to avoid catabolite repression). T h e newly injected lambda D N A circularizes at its cohesive (cos) termini, which are composed of complementary, 12nucleotide 5' single-strand protrusions. A host D N A ligase seals the nicks flanking the cos sequence, and the D N A is supercoiled by a host gyrase (Figure 2 - 2 ) .
Preparations of the arms are ligated in the presence of restriction overhang-compatible inserts to form concatenated molecules (Figure 1-3). Although many permutations of arms and inserts are possible, the combination of an insert flanked by left and right arms can form a packageable infectious g e n o m e . T h e major advantage of lambda cloning over other vector cloning is in vitro D N A packaging. T h e in vitro packaging reaction is surprisingly efficient. 88 x 10 lambda genomes. 88 x 1 0 infectious phage particles per micro8 gram of lambda D N A .