E. coli Plasmid Vectors: Methods and Applications / Edition 1

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Overview

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 prools 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.

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Editorial Reviews

From the Publisher
...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

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Product Details

  • ISBN-13: 9781617373916
  • Publisher: Springer-Verlag New York, LLC
  • Publication date: 11/10/2010
  • Series: Methods in Molecular Biology Series , #235
  • Edition description: Softcover reprint of hardcover 1st ed. 2003
  • Edition number: 1
  • Pages: 316
  • Product dimensions: 9.00 (w) x 6.00 (h) x 0.74 (d)

Table of Contents

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 by Inverse 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

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