Cloning into Bacteriophage M13 Vectors Protocol Protocol describes three standard methods to construct bacteriophage M13 recombinants: (1) ligating insert DNA to a linearized vector, prepared by cleavage of M13 RF with a single restriction enzyme; (2) using alkaline phosphatase to suppress self-ligation of the linearized vector, and (3) using M13 RF cleaved with two restriction enzymes for directional cloning.
Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA Protocol Protocol used chiefly to generate large stocks of double-stranded DNA of strains of M13 that are routinely used as cloning vectors. Large amounts of single-stranded DNA of an individual recombinant may occasionally be needed for specific purposes, e.g., to generate many preparations of a particular radiolabeled probe or to construct large numbers of site-directed mutants.
Preparation of Single-stranded Bacteriophage M13 DNA Protocol Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected cells into the surrounding medium. The filamentous particles are concentrated by precipitation from a high-ionic-strength buffer with polyethylene glycol. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. This protocol is generally used to prepare single-stranded DNA from a small number of M13 isolates.
Preparation of Uracil-containing Single-stranded Bacteriophage M13 DNA Protocol Single-stranded templates of bacteriophage M13 DNA containing 20-30 residues of uracil in place of thymine are generated during growth of the bacteriophage in an F' strain of E. coli carrying mutations in the ung and dut genes. This DNA is used as a template in the Kunkel method of oligonucleotide-directed mutagenesis (Oligonucleotide-directed Mutagenesis of Single-stranded DNA).
Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligos Plaques formed by M13 bacteriophages or bacterial colonies transformed by plasmids carrying specific mutations can be detected by hybridization, using a radiolabeled oligonucleotide that forms a perfect duplex with the mutant sequence. Hybridization is carried out under conditions of low stringency that allow the radiolabeled oligonucleotide to anneal to both mutant and wild-type DNAs.
Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates Protocol A synthetic oligonucleotide annealed to single-stranded DNA derived from a recombinant bacteriophage M13 or phagemid template is used to prime the synthesis of complementary radiolabeled DNA. Synthesis is catalyzed by the Klenow fragment of E. coli DNA polymerase I, which extends the annealed primer for various distances along the single-stranded template DNA.
Synthesis of Single-stranded DNA Probes of Heterogeneous Length from Bacteriophage M13 Templates Technique yields a heterogeneous population of short radiolabeled molecules 200-300 nucleotides in length. These probes are synthesized, as in Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates, by extension of an oligonucleotide primer on a single-stranded DNA template. The radiolabeled products of the reaction are then separated from the template by electrophoresis through a denaturing gel from which they are eluted directly into hybridization buffer.
