E. Coli Plasmid Vectors: Methods and Applications

ISBN-13: 9781588291516 / Angielski / Twarda / 2003 / 316 str.

Nicola Casali; Andrew Preston; Nicola Casali

Opis

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.

Recenzje

...a scientific review and a methodological "cook-book" of high quality. It is truly a highly practical handbook, useful for all scientists working with gene cloning and manipulation." - Folia Microbiologica

"...a very useful addition to laboratories investigating plasmids per se, and for those employing plasmids as vectors for gene cloning." - Microbiology Today

Spis treści

The Function and Organization of Plasmids Finbarr 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 and Wade A. Nichols Isolation of Plasmids from E. coli by Alkaline Lysis Sabine Ehrt and Dirk Schnappinger Isolation of Plasmids from E. coli by Boiling Lysis Sabine Ehrt and Dirk Schnappinger High-Purity Plasmid Isolation Using Silica Oxide Stefan Grimm and Frank Voß-Neudecker High-Throughput Plasmid Extraction Using Microtiter Plates Michael A. Quail Isolation of Cosmid and BAC DNA from E. coli Daniel Sinnett and Alexandre Montpetit 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 and Sharon Kendall Extraction of DNA from Agarose Gels Nicholas Downey Cloning PCR Products with T-Vectors Wade A. Nichols Construction of Genomic Libraries in l-Vectors Yilun Wang, Zheng Cao, Darryl Hood, and James G. Townsel Rapid Screening of Recombinant Plasmids Sangwei Lu Restriction Analysis of Recombinant Plasmids Joanne Goranson-Siekierke and Jarrod L. Erbe Screening Recombinant DNA Libraries Wade A. Nichols Sequencing Using Fluorescent-Labeled Nucleotides Allison F. Gillaspy Site-Directed Mutagenesis Using the Megaprimer Method Zhidong Xu, Alessia Colosimo, and Dieter C. Gruenert Site-Directed Mutagenesis byInverse PCR Clifford N. Dominy and David W. Andrews Creating Nested DNA Deletions Using Exonuclease III Rosamund Powles and Lafras M. Steyn Transposon and Transposome Mutagenesis of Plasmids, Cosmids, and BACs Alistair McGregor In Vitro Transcription and Translation Farahnaz Movahedzadeh, Susana González Rico, and Robert A. Cox 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 Index

Blurb

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.