Abstract

Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M 13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.

Author Keywords: Recombinant DNA; molecular cloning; polycloning sites; progressive deletions

Abbreviations: Ac, activator; Ap, ampicillin; B-broth, Bactotryptone broth; Cm, chloramphenicol; Δ, deletion; DTT, dithothreitol; EMS, ethylmethane sulfonate; Exo III and VII, exonuclease III and VII; HA, hydroxylamine hydrochloride; IPTG, isopropyl-β- -thiogalactopyranoside; LB, Luria broth; M13UC, see RESULTS, section c2; moi, multiplicity of infection; pfu, plaque-forming units; PHS, primer hybridization site; ^R, resistance; RF, replicative form; RT, room temperature; Sm, streptomycin; STE, 10 mM NaCl, 10 mM Tris · HCL pH 7.5, 1 mM EDTA; Tc, tetracycline; Xgal, 5-bromo-4-chloro-indolyl-β- -galactopyranoside; YT, yeast tryptone; [], indicates plasmidcarrier state; Δ, deletion

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