Molecular Biology Techniques

Molecular Biology Techniques

A Classroom Laboratory Manual

3rd Edition - October 18, 2011

Write a review

  • Authors: Heather Miller, D. Scott Witherow, Sue Carson
  • Paperback ISBN: 9780123855442
  • eBook ISBN: 9780123855459

Purchase options

Purchase options
Available
DRM-free (PDF, EPub, Mobi)
Sales tax will be calculated at check-out

Institutional Subscription

Free Global Shipping
No minimum order

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

  • 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

Product details

  • No. of pages: 232
  • Language: English
  • Copyright: © Academic Press 2011
  • Published: October 18, 2011
  • Imprint: Academic Press
  • Paperback ISBN: 9780123855442
  • eBook ISBN: 9780123855459

About the Authors

Heather Miller

Heather Miller
Dr. Heather Miller is an Assistant Professor of Biochemistry in the Chemistry Department at High Point University (High Point, NC). She graduated from Clarion University of Pennsylvania (Clarion, PA) with a B.S. in Molecular Biology/Biotechnology, and from Duke University (Durham, NC) with a Ph.D. in Molecular Genetics and Microbiology. She completed a teaching postdoctoral position in the Biotechnology Program at North Carolina State University (Raleigh, NC). Her area of scientific expertise is RNA biology. Her research focuses on HIV-1 gene expression and the coupling of transcription and splicing in humans. She has taught at the college level for nine years, and is engaged in the scholarship of teaching and learning.

Affiliations and Expertise

Department of Chemistry, High Point University, High Point, NC USA

D. Scott Witherow

D. Scott Witherow
Dr. Witherow graduated from Rollins College (Winter Park, FL) with an A.B. in Chemistry, and from the University of Miami (Miami, FL) with a Ph.D. in Molecular and Cellular Pharmacology. His research has focused primarily on G protein-mediated signal transduction processes in mammalian systems. Following two research postdoctoral fellowships, Dr. Witherow served as a teaching postdoctoral fellow at North Carolina State University, where he published and presented multiple papers in the field of science education and developed a passion for teaching. He is currently an Associate Professor at The University of Tampa, where he has been teaching undergraduate students since 2011.

Affiliations and Expertise

Department of Chemistry, Biochemistry, and Physics, The University of Tampa, Tampa, FL USA

Sue Carson

Sue Carson

Affiliations and Expertise

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

Ratings and Reviews

Write a review

There are currently no reviews for "Molecular Biology Techniques"