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 third 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 PR

Details

No. of pages:
232
Language:
English
Copyright:
© 2012
Published:
Imprint:
Academic Press
Print ISBN:
9780123855442
Electronic ISBN:
9780123855459

About the authors

Heather Miller

Affiliations and Expertise

North Carolina State University, Raleigh, USA

D. Scott Witherow

Affiliations and Expertise

North Carolina State University, Raleigh, USA

Reviews

"Overall, this manual represents an invaluable training material on practical molecular biology for undergraduates, graduates, and inexperienced researchers. It could also introduce more experienced researchers to experiments that they have not considered previously."--Science Progress, vol 95, issue 2, 2012
"Whilst molecular biology has been the focus of course curricula in various bioscience educational programmes, there has been a lack of well-designed laboratory manuals to recommend for the practical sessions of these courses. The third edition of ‘Molecular Biology Techniques’ is one such excellent classroom laboratory manual. It encompasses experiments for 19 laboratory sessions presented as a semester-long project that gets students involved in a comprehensive experimental story from gene cloning to protein purification. The authors have employed the versatility of the PCR technique in various experiments and have also taken advantage of the enhanced green fluorescent protein in visualising positive clones. A new section involving five laboratory sessions on measuring mRNA levels has been added to this third edition. Overall, this manual represents an invaluable training material on practical molecular biology for undergraduates, graduates, and inexperienced researchers. It could also introduce more experienced researchers to experiments that they have not considered previously."--Science Progress