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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.
- 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
- 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
- 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
- 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
- Chapter 5. Carrying Out Q-PCR
- 5.1. Introduction
- 5.2. Reagents
- 5.3. Basic Protocols
- 5.4. Absolute Quantification
- 5.5. Relative Quantification
- 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
- 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
- Appendix 1
- Appendix 2
- No. of pages:
- © Academic Press 2016
- 26th October 2016
- Academic Press
- eBook ISBN:
- Paperback ISBN:
Sarah Maddocks, PhD, is a lecturer in Microbiology at Cardiff Metropolitan University, UK. Her research focuses on understanding the interactions between pathogens and their host during the infection process. The overarching aim of these studies is to understand how pathogens utilise or disrupt host processes and to explore and exploit novel ways of impairing these host-pathogen interactions to facilitate clearance of infection. Dr. Maddocks’ research also includes biofilm and co-culture models, genomic and molecular analysis of gene expression to prove clinically relevant hypotheses.
Lecturer in Microbiology at Cardiff Metropolitan University, UK
Rowena Jenkins, PhD, is a lecturer in Microbiology and Infectious Disease in the College of Medicine at Swansea University, UK. Her research team are focused on the effect of novel antimicrobial agents on pathogenic bacteria found in clinical infections, particularly chronic infections such as those seen in diabetic foot ulcers and cystic fibrosis lungs infection, and the impact on the microbiome within those conditions. Dr. Jenkins also has an interest in the potential for natural antimicrobial agents to improve the efficacy of conventional antibiotics against antibiotic resistant bacteria and biofilms.
Lecturer in Microbiology and Infectious Disease, College of Medicine at Swansea University, UK
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