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Molecular Biology Techniques

A Classroom Laboratory Manual

  • 3rd Edition - October 18, 2011
  • Newer edition is available
  • Authors: Heather B. Miller, D. Scott Witherow, Sue Carson
  • Language: English

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 ex… Read more

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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 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

Review quotes

"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

Product details

About the authors

HM

Heather B. Miller

Dr. Heather B. Miller is an Associate Professor of Biochemistry at High Point University (High Point, NC). Her area of scientific expertise is RNA biology. Her research focuses on MRSA and other pathogens’ gene expression when challenged with antibiotics and novel antibiotic adjuvants. She has mentored over 30 undergraduate students and is the Principal Investigator of a National Institutes of Health (NIH) Academic Research Enhancement Award (AREA). She was named a Henry Dreyfus Teacher-Scholar for her outstanding accomplishments in research and education in the chemical sciences. She has developed and taught multiple biochemistry and biotechnology courses and has published and presented a number of peer-reviewed papers in the scholarship of teaching and learning. Dr. Miller graduated from Clarion University of Pennsylvania (Clarion, PA) with a BS in Molecular Biology/Biotechnology, and from Duke University (Durham, NC) with a PhD in Molecular Genetics and Microbiology. She completed a teaching postdoctoral position in the Biotechnology Program at North Carolina State University
Affiliations and expertise
Department of Chemistry, High Point University, High Point, NC, USA

DW

D. Scott Witherow

Dr. D. Scott Witherow is an Associate Professor of Biochemistry at The University of Tampa (Tampa, FL). His research focuses primarily on the regulation of G-protein-mediated signal transduction processes and biochemical education and pedagogy. In addition to mentoring over 20 undergraduate research students, Dr. Witherow has designed and published biochemistry lab courses and has been recognized by the American Society for Biochemistry and Molecular Biology as an education fellow for his efforts in the areas of instruction and assessment. He graduated from Rollins College (Winter Park, FL) with an AB in Chemistry and from the University of Miami (Miami, FL) with a PhD in Molecular and Cellular Pharmacology. Following traditional postdoctoral research positions at Duke University, he transitioned into pedagogy through a teaching postdoctoral position at North Carolina State University
Affiliations and expertise
Department of Chemistry and Biochemistry, The University of Tampa, Tampa, FL USA

SC

Sue Carson

Dr. Susan Carson is a Professor of Plant and Microbial Biology at North Carolina State University (Raleigh, NC) where she has served on the faculty since 2001. She directs the Master of Microbial Biotechnology Program and also leads a university-wide faculty development program focused on enhancing students’ critical and creative thinking skills across disciplines. Prior to her current role, Dr. Carson spent over a decade leading curriculum development for the North Carolina State Biotechnology Program and two years as a Program Officer at the National Science Foundation Division of Undergraduate Education. Her current work focuses on college-level biology education, enhancing students’ higher order thinking across disciplines, and integration of Design Thinking in a Professional Science Masters (PSM) program. She graduated from Rutgers University (New Brunswick, NJ) with a BS in Biotechnology, and from the University of North Carolina (Chapel Hill, NC) with a PhD in Microbiology.

Affiliations and expertise
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, USA

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