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Molecular Biology Techniques,

Edition 3 A Classroom Laboratory Manual

Editors: By Heather B. Miller, D. Scott Witherow and Sue Carson

Publication Date: 07 Nov 2011

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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

About the author

By Heather B. Miller, Department of Chemistry, High Point University, High Point, NC USA; D. Scott Witherow, Department of Chemistry, Biochemistry, and Physics, The University of Tampa, Tampa, FL USA and Sue Carson, North Carolina State University, Raleigh, U.S.A.

Preface

About the Authors

Acknowledgements

Note to Instructors

Instrumentation

Nomenclature

Introduction

Part 1. Manipulation of DNA

Lab Session 1. Getting Oriented

Lab Session 2. Purification and Digestion of Plasmid (Vector) DNA

Lab Session 3. PCR Amplification of egfp and Completion of Vector Preparation

Lab Session 4. Preparation of Insert DNA (egfp) PCR Product

Lab Session 5. DNA Ligation and Transformation of Escherichia coli

Part 2. Screening Transformants

Lab Session 6. Colony Hybridization

Lab Session 6A. Interim Laboratory Session

Lab Session 6B. Colony Hybridization: Monoclonal Antibody Probe

Lab Session 7. Characterization of Recombinant Clones

Lab Session 7A. Completion of Colony Hybridization with a Monoclonal Antibody Probe

Lab Session 7B. PCR Screening

Lab Session 7C. Prepare Fresh Replica Plate

Lab Session 8. Characterization of Recombinant Clones

Lab Session 8A. Interim Laboratory Session

Lab Session 8B. Analysis of PCR Screen Results

Lab Session 8C. Isolation of Miniprep DNA from Potential Transformants

Lab Session 8D. Visualization of Green Fluorescent Protein: Part 1

Lab Session 9. Characterization of Recombinant Clones

Lab Session 9A. Characterization of Miniprep DNA from Potential Transformants (Restriction Enzyme Analysis of Putative Transformants)

Lab Session 9B. Visualization of Green Fluorescent Protein: Part 2

Lab Session 9C. Computational Analysis of DNA Sequence from a Positive Clone: Part 1

Lab Session 10. Computational Analysis of DNA Sequence from a Positive Clone

Part 3. Expression, Detection and Purification of Recombinant Proteins from Bacteria

Lab Session 11. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot

Lab Session 11A. Interim Laboratory Session

Lab Session 11B. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot

Lab Session 12. Expression of Fusion Protein from Positive Clones, SDS-PAGE and Western Blot

Lab Session 13. Extraction of Recombinant Protein from Escherichia coli Using a Glutathione Affinity Column

Lab Session 13A. Interim Laboratory Session

Lab Session 13B. Extraction of Recombinant Protein from Escherichia coli and Purification Using a Glutathione Affinity Column

Lab Session 14. Analysis of Purification Fractions

Lab Session 14A. Analysis of Purification Fractions

Lab Session 14B. Replica Plate Positive Clone

Part 4. Analysis of mRNA Levels

Lab Session 15. Total RNA Purification

Lab Session 15A. Interim Laboratory Session

Lab Session 15B. Total RNA Purification

Lab Session 16. Analysis of gst::egfp mRNA Levels by RT-qPCR

Lab Session 17. Analysis of gst::egfp mRNA Levels by RT-qPCR

Lab Session 18. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR

Lab Session 19. Analysis of gst::egfp mRNA Levels by Semi-Quantitative RT-PCR

Appendix 1. Equipment

Appendix 2. Prep List

Appendix 3. Preparation of Competent E. coli Cells

Appendix 4. Pre-Lab Questions

Index

Title 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, 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

ISBN: 9780123855442

Page Count: 232

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