Title E. coli plasmid vectors : methods and applications / edited by Nicola Casali and Andrew Preston

Published Totowa, N.J. : Humana Press, ©2003

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Description 1 online resource (xi, 316 pages) : illustrations Series Methods in molecular biology ; v. 235 Methods in molecular biology (Clifton, N.J.) ; v. 235 Contents The function and organization of plasmids / Finbar Hayes -- Choosing a cloning vector / Andrew Preston -- Escherichia coli host strains / Nicola Casali -- Chemical transformation of E. coli / W. Edward Swords -- Electroporation of E. coli / Claire A. Woodall -- DNA transfer by bacterial conjugation / Claire A. Woodall -- Cosmid packaging and infection of E. coli / Mallory J.A. White -- Isolation of plasmids from E. coli by alkaline lysis / Sabine Ehrt -- Isolation of plasmids from E. coli by boiling lysis / Sabine Ehrt -- High-purity plasmid isolation using silica oxide / Stefan Grimm -- High-throughput plasmid extraction using microtiter plates / Michael A. Quail -- Isolation of cosmid and BAC DNA from E. coli / Daniel Sinnett -- Preparation of single-stranded DNA from phagemid vectors / W. Edward Swords -- Using desktop cloning software to plan, track, and evaluate cloning projects / Robert H. Gross -- Cloning in plasmid vectors / Carey Pashley -- Extraction of DNA from agarose gels / Nicholas Downey -- Cloning PCR products with T-vectors / Wade A. Nichols -- Construction of genomic libraries in v-vectors / Yilun Wang -- Rapid screening of recombinant plasmids / Sangwei Lu -- Restriction analysis of recombinant plasmids / Joanne Goranson-Siekierke -- Screening recombinant DNA libraries / Wade A. Nichols -- Sequencing using fluorescent-labeled nucleotides / Allison F. Gillaspy -- Site-directed mutagenesis using the megaprimer method / Zhidong Xu -- Site-directed mutagenesis by inverse PCR / Clifford N. Dominy -- Creating nested DNA deletions using exonuclease III / Rosamund Powles -- Transposon and transposome mutagenesis of plasmids, cosmids, and BACs / Alistair McGregor -- In vitro transcription and translation / Farahnaz Movahedzadeh -- Vectors for the expression of recombinant proteins in E. coli / Sally A. Cantrell -- Expression of recombinant proteins from lac promoters / Charles R. Sweet -- Plasmid-based reporter genes: assays for B-galactosidase and alkaline phosphatase activities / Minghsun Liu -- Plasmid-based reporter genes: assays for green fluorescent protein / Sergei R. Doulatov Summary Manipulation of recombinant DNA, which is almost exclusively performed using the host E. coli, constitutes one of the fundamental methodologies of molecular biotechnology. In E. coli Plasmid Vectors, experienced bench researchers describe their proven techniques for the manipulation of recombinant plasmids utilizing this popular bacterial host. The authors describe readily reproducible 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. Also presented are methods for common downstream applications, such as mutagenesis, expression of recombinant proteins and RNA transcripts, and uses of reporter genes. Each fully tested protocol is described in step-by-step detail by an established expert in the field and includes an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, tips on troubleshooting and avoiding known pitfalls and, where needed, a discussion of the interpretation and use of the results. Comprehensive and highly practical, E. coli Plasmid Vectors offers those new to the field a basic guide to the use of plasmid vectors in the cloning host E. coli, and those more experienced researchers a broad-ranging, proven array of successful techniques Bibliography Includes bibliographical references and index Notes English Print version record Subject Plasmids -- Laboratory manuals Escherichia coli -- Laboratory manuals Genetic vectors -- Laboratory manuals Recombinant DNA. Escherichia coli -- genetics Plasmids -- genetics Cloning, Molecular -- methods DNA, Recombinant SCIENCE -- Life Sciences -- Genetics & Genomics. Recombinant DNA Escherichia coli Genetic vectors Plasmids Genre/Form Laboratory manuals Laboratory manuals. Manuels de laboratoire. Form Electronic book Author Casali, Nicola Preston, Andrew, 1969- ISBN 9781592594092 1592594093 1280842822 9781280842825 9786610842827 6610842825

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