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agarose gel aliquots bacteria bacteriophage M13 bacteriophage particles bacteriophage T4 DNA BamHI buffer carrying cDNA cells centrifugation at 12,000g Cl pH cleavage cleaved cloning cohesive termini coli DNA polymerase containing cosmid cosmid vector culture digestion DNA fragments DNA ligase DNA molecules DNA polymerase double-stranded DNA EcoRI EDTA efficiently Escherichia coli ethanol ethidium bromide eukaryotic eukaryotic DNA exonuclease extracts final concentration foreign DNA gel electrophoresis gene genetic Hindlll hours at 37°C hybridization Incubate infected inserted intergenic region ligation reaction method methylation microfuge tube minutes at 4°C minutes at room mutations Natl nitrocellulose nitrocellulose filter Nucleic Acids Nucleic Acids Res nucleotides origin of replication packaging pellet phagemid plaques plasmid plasmid DNA polycloning prepared probes protein purified recA recombinant remove restriction enzymes room temperature segment of foreign single-stranded DNA solution sterile stored strains strand supernatant T4 DNA ligase template teriophage Transfer vector DNA vitro volume xg/ml