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Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors |
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| Authors: | C Yanisch-Perron J Vieira J Messing |
| Institution: | Department of Biochemistry, University of Minnesota, St. Paul. MN 55108 U.S.A. Tel. (612) 376 1509 |
| 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 M13mp 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. |
| Keywords: | Recombinant DNA molecular cloning polycloning sites progressive deletions 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 LB Luria broth M13UC see RESULTS section c2 moi multiplicity of infection pfu plaque-forming units PHS primer hybridization site 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 YT yeast tryptone [] indicates plasmidcarrier state Δ deletion |
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