Manipulation and Expression of Recombinant DNA

Manipulation and Expression of Recombinant DNA

A Classroom Laboratory Manual

2nd Edition - December 6, 2005

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  • Authors: Sue Carson, Dominique Robertson
  • eBook ISBN: 9780080456546

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Description

This manual is an indispensable tool for introducing advanced undergraduates and beginning graduate students to the techniques of recombinant DNA technology, or gene cloning and expression. The techniques used in basic research and biotechnology laboratories are covered in detail. Students gain hands-on experience from start to finish in subcloning a gene into an expression vector, through purification of the recombinant protein.The second edition has been completely re-written, with new laboratory exercises and all new illustrations and text, designed for a typical 15-week semester, rather than a 4-week intensive course. The “project” approach to experiments was maintained: students still follow a cloning project through to completion, culminating in the purification of recombinant protein. It takes advantage of the enhanced green fluorescent protein—students can actually visualize positive clones following IPTG induction.

Key Features

*Cover basic concepts and techniques used in molecular biology research labs
*Student-tested labs proven successful in a real classroom laboratories
*Exercises simulate a cloning project that would be performed in a real research lab
*"Project" approach to experiments gives students an overview of the entire process
*Prep-list appendix contains necessary recipes and catalog numbers, providing staff with detailed instructions

Readership

Graduate and undergraduate students studying biochemistry, molecular biology, biotechnology, and cell biology

Table of Contents

  • CONTENTS

    Foreword
    Acknowledgements
    Note to Instructors
    Instrumentation
    Nomenclature

    INTRODUCTION: Conceptual outline for experiments

    PART I MANIPULATION OF DNA

    Lab session 1: Getting oriented; Practicing with pipetmen
    Lab session 2: Large scale purification of plasmid DNA
    Lab session 3: Preparation of expression vector DNA (pET-41a(+), a GST fusion protein vector)
    Lab session 4: Preparation of insert DNA (egfp)
    Lab session 5: Preparation of transformation-competent cells and control transformation
    Lab session 6: DNA ligation and transformation of Escherichia coli

    PART II SCREENING TRANSFORMANTS


    Lab session 7: Colony hybridizations
    Lab session 7a: Interim laboratory session:
    Lab session 7b: Colony hybridization: DNA probe
    Lab session 7c: Colony hybridization: Monoclonal antibody probe
    Lab session 8: Completion of colony hybridization with DNA probe
    Lab session 9: Characterization of recombinant clones
    Lab session 9a: Completion of colony hybridization with mAB probe
    Lab session 9b: PCR screen
    Lab session 9c: Visualization of green fluorescent protein: Part 1
    Lab session 10: Further characterization of recombinant clones
    Lab session 10a: Interim laboratory session:
    Lab session 10b: Analysis of PCR screen results
    Lab session 10c: Isolation and characterization of miniprep DNA from potential transformants (Restriction analysis of putative transformants)
    Lab session 10d: Visualization of green fluorescent protein: Part 2

    PART III EXPRESSION, DETECTION, AND PURIFICATION OF RECOMBINANT PROTEINS FROM BACTERIA


    Lab session 11: Expression of fusion protein from positive clones and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Immunological analysis (Western blot): Part 1
    Lab session 12: Expression of fusion protein from positive clones and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Immunological analysis (Western blot): Part 2
    Lab session 13: Extraction of recombinant protein from Escherichia coli using a glutathione affinity column
    Interim laboratory session: I. Laboratory exercise: Inoculate cultures for protein purification
    Lab session 14: Analysis of purification fractions
    Appendices: Equipment, Prep list, Making sense of orientation

Product details

  • No. of pages: 176
  • Language: English
  • Copyright: © Academic Press 2005
  • Published: December 6, 2005
  • Imprint: Academic Press
  • eBook ISBN: 9780080456546

About the Authors

Sue Carson

Sue Carson

Affiliations and Expertise

North Carolina State University, Raleigh, U.S.A.

Dominique Robertson

Affiliations and Expertise

North Carolina State University, Raleigh, U.S.A.

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