E. coli Plasmid Vectors Methods and Applications

A comprehensive collection of readily reproducible techniques for the manipulation of recombinant plasmids using the bacterial host E. coli. The authors describe proven methods for cloning DNA into plasmid vectors, transforming plasmids into E. coli, and analyzing recombinant clones. They also include protocols for the construction and screening of libraries, as well as specific techniques for specialized cloning vehicles, such as cosmids, bacterial artificial chromosomes, l vectors, and phagemids. Common downstream applications such as mutagenesis of plasmids, recombinant protein expression, and the use of reporter genes, are also described.

The Function and Organization of Plasmids.- Choosing a Cloning Vector.- Escherichia coli Host Strains.- Chemical Transformation of E. coli.- Electroporation of E. coli.- DNA Transfer by Bacterial Conjugation.- Cosmid Packaging and Infection of E. coli.- Isolation of Plasmids from E. coli by Alkaline Lysis.- Isolation of Plasmids from E. coli by Boiling Lysis.- High-Purity Plasmid Isolation Using Silica Oxide.- High-Throughput Plasmid Extraction Using Microtiter Plates.- Isolation of Cosmid and BAC DNA from E. coli.- Preparation of Single-Stranded DNA from Phagemid Vectors.- Using Desktop Cloning Software to Plan, Track, and Evaluate Cloning Projects.- Cloning in Plasmid Vectors.- Extraction of DNA from Agarose Gels.- Cloning PCR Products with T-Vectors.- Construction of Genomic Libraries in ?-Vectors.- Rapid Screening of Recombinant Plasmids.- Restriction Analysis of Recombinant Plasmids.- Screening Recombinant DNA Libraries.- Sequencing Using Fluorescent-Labeled Nucleotides.- Site-Directed Mutagenesis Using the Megaprimer Method.- Site-Directed Mutagenesis by Inverse PCR.- Creating Nested DNA Deletions Using Exonuclease III.- Transposon and Transposome Mutagenesis of Plasmids, Cosmids, and BACs.- In Vitro Transcription and Translation.- Vectors for the Expression of Recombinant Proteins in E. coli.- Expression of Recombinant Proteins From lac Promoters.- Plasmid-Based Reporter Genes.- Plasmid-Based Reporter Genes.