Understanding PCR - 1st Edition - ISBN: 9780128026830, 9780128026977

Understanding PCR

1st Edition

A Practical Bench-Top Guide

Authors: Sarah Maddocks Rowena Jenkins
eBook ISBN: 9780128026977
Paperback ISBN: 9780128026830
Imprint: Academic Press
Published Date: 26th October 2016
Page Count: 98
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Understanding PCR: A Practical Bench-Top Guide gives you all of the information you need to plan your first PCR, from reagents to conditions to analysis and beyond. It is a user friendly book that has step-by-step basic protocols, which can be adapted to your needs. Includes helpful information such as where to order your reagents and basic troubleshooting hints and tips.

Key Features

  • Includes resources for reagents
  • Explains basic laboratory preparation
  • Provides straightforward experimental protocols
  • Incorporates fundamental analytical techniques
  • Contains a troubleshooting guide


Researchers, lab professionals, and students in molecular biology

Table of Contents

  • Author Biographies
  • Chapter 1. Things to Consider Before Preparing to Undertake Your First Polymerase Chain Reaction
    • 1.1. Introduction
    • 1.2. Basic Laboratory Preparation
    • 1.3. Maintaining a Nuclease-Free Environment
    • 1.4. Consideration of Timescales
    • 1.5. General Protocol for Extracting DNA
    • 1.6. What Reagents You Will Need and Where to Buy Them
    • 1.7. Safety Considerations
    • Summary
  • Chapter 2. Designing and Ordering Your Polymerase Chain Reaction Primers
    • 2.1. Introduction
    • 2.2. Choosing a “Template”
    • 2.3. Finding Gene Sequences Using Online Databases
    • 2.4. Identifying Your Amplicon of Interest
    • 2.5. Contending With Introns and Exons
    • 2.6. Basic Primer Parameters
    • 2.7. Calculating Annealing and Melting Temperatures
    • 2.8. Tools for Primer Design
    • 2.9. Ordering Your Primers
    • 2.10. Reconstituting Primers and Preparing a Working Stock
    • 2.11. Testing a New Primer Pair
    • 2.12. Common Problems and Troubleshooting
  • Chapter 3. Analyzing Your Template DNA and/or PCR Product
    • 3.1. Introduction
    • 3.2. Basic DNA/PCR Analysis
    • 3.3. “Running” an Agarose Gel
    • 3.4. Staining and Visualizing DNA
    • 3.5. Which Molecular Weight Marker is Best for You?
    • 3.6. Imaging Your Gel and Interpreting What It Shows
    • 3.7. Quantifying DNA/PCR Product Using Electrophoresis
    • Summary
  • Chapter 4. Quantitative PCR: Things to Consider
    • 4.1. Introduction
    • 4.2. What are You Trying to Achieve?
    • 4.3. The Importance of Housekeeping
    • 4.4. What Q-PCR Machine Should You Use, or What Machine Is Available to You?
    • 4.5. Efficiency Parameters
    • 4.6. Linearity and Amplification Efficiency Based in the Template Material
    • 4.7. Amplification Efficiency
    • 4.8. cDNA Conversion and Quantification
    • 4.9. Planning to Use Q-PCR for Multiplex
    • Summary
  • Chapter 5. Carrying Out Q-PCR
    • 5.1. Introduction
    • 5.2. Reagents
    • 5.3. Basic Protocols
    • 5.4. Absolute Quantification
    • 5.5. Relative Quantification
    • Summary
  • Chapter 6. Using PCR for Cloning and Protein Expression
    • 6.1. Introduction
    • 6.2. Enzymes for Cloning
    • 6.3. “Cleaning” PCR Products for Cloning
    • 6.4. Directional Cloning
    • 6.5. Ligation of PCR Product and Vector
    • 6.6. Transforming and Analyzing the Plasmid Construct
    • 6.7. Sequencing Plasmid Constructs
    • Summary
  • Chapter 7. Polymerase Chain Reaction for Knocking Out Genes
    • 7.1. Introduction
    • 7.2. How to Knockout a Gene Using PCR
    • 7.3. Transferring Engineered PCR Fragments into the Target Organism Using a Suicide Vector
    • 7.4. The λ Red Recombinase System
    • 7.5. Screening for Knockout Mutations
    • Summary
  • Appendix 1
  • Appendix 2
  • Index


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© Academic Press 2017
Academic Press
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About the Author

Sarah Maddocks

Dr Sarah E. Maddocks is a Lecturer of Microbiology at Cardiff Metropolitan University, UK. She received a PhD from the University of Reading in 2005 and worked on bacterial virulence before embarking upon investigations into the host-pathogen relationship during infection. Her main research interests involve the study of wound colonisation, bacterial persistence and the shift from colonisation to infection.

Affiliations and Expertise

Associate Professor, Cardiff Metropolitan University, UK

Rowena Jenkins

Rowena Jenkins is a Lecturer in Microbiology at Cardiff Metropolitan University, from which she received her MSc in Biomedical Science and Medical Microbiology and PhD, and a Fellow of the Higher Education Academy. Her main research has focused on the antimicrobial effects of Manuka honey on potentially pathogenic microorganisms in healthcare and wound management. Cellular morphology, physiology, biofilm prevention/disruption, adhesion/invasion, virulence expression and proteomic/genetic expression profiles of organisms such as MRSA and P. aeruginosa have been studied in response to varying treatments with honey. The data generated by these in vitro studies help reveal how antimicrobial agents might be applied in clinical settings. Rowena is also exploring how novel antimicrobial agents can potentiate antibiotics already in use but whose effectiveness is now challenged by antimicrobial resistance.

Affiliations and Expertise

Professor, Cardiff Metropolitan University, UK

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