PCR Guru: An Ultimate Benchtop Reference for Molecular Biologists
By Ayaz Najafov and Gerta Hoxhaj
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About this ebook
PCR Guru: An Ultimate Benchtop Reference for Molecular Biologists is provides researchers in molecular biology with a handy reference for approaching and solving challenging problems associated with PCR setup and optimization. As a laboratory guide, it emphasizes the technical aspects of employing PCR as a tool in molecular biology laboratories. The book covers the history of PCR and the basic science underlying it. It then discusses PCR at the bench level, starting with detailed description and tips on primer design, and continuing with the standard protocols used to perform PCR.
- Provides troubleshooting tips for various types of modifications of standard protocols
- Contains unique “Good Practices and Tips that are indispensable for the beginner and expert alike
- Features “Special Cases with applications of PCR, optimization, and troubleshooting
- Includes detailed appendices with tables, figures, and key protocols
- Organized as a systematic, concentrated resource to save time when addressing a PCR problem
Ayaz Najafov
Ayaz was born in Baku, Azerbaijan. He obtained his BSc and MSc degrees in Molecular Biology and Genetics from Bosphorus University, Istanbul, Turkey and got a 2-year training in Biochemistry as an intern at Baylor College of Medicine, Houston, Texas, USA. Ayaz pursued his PhD in Biochemistry at MRC Protein Phosphorylation Unit at Dundee University, Scotland, UK in Prof. Dario Alessi’s lab. Ayaz is currently a postdoctoral fellow in Prof. Junying Yuan’s lab at Harvard Medical School, Cell Biology Department.
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Book preview
PCR Guru - Ayaz Najafov
PCR Guru
An Ultimate Benchtop Reference for Molecular Biologists
Ayaz Najafov
Harvard Medical School, Department of Cell Biology, Boston, MA, United States
Gerta Hoxhaj
Harvard T.H. Chan School of Public Health, Department of Genetics and Complex Diseases, Boston, MA, United States
Table of Contents
Cover
Title page
Copyright
Dedications
Biography
Preface
Acknowledgments
Note to the Reader
Chapter 1: Introduction
Abstract
1.1. A bit of history
1.2. What is PCR?
1.3. PCR components
1.4. Exponential nature of PCR
1.5. Popularity of PCR
Chapter 2: Procedure
Abstract
2.1. Primer design
2.2. Standard PCR setup
2.3. Controls
2.4. Cycling parameters
Chapter 3: Good PCR Practices
Abstract
Chapter 4: Optimization and Troubleshooting
Abstract
4.1. Optimization rules
4.2. General optimization strategies
4.3. Troubleshooting specific problems
Chapter 5: Tips and Tricks
Abstract
Chapter 6: Special Cases
Abstract
Glossary and Abbreviations
Appendix A: Agarose Gel Electrophoresis Protocol
Appendix B: Silver Staining of DNA Gels
Appendix C: Plasmid Miniprep Protocol (by using QIAprep Spin Miniprep
Kit)
Appendix D: Restriction Enzyme Digestion Protocol
Appendix E: Protocol for DNA Concentration Estimation Using Hoechst 33258
Appendix F: Formulas
Appendix G: Common Primers for Sequencing, Promoters, Housekeeping Genes, and Reporters
Appendix H: Tags
Appendix I: Thermostable DNA polymerases
Appendix J: Example of a Daily PCR Log
Appendix K: Common Buffers and Solutions
Appendix L: Useful Numbers
Appendix M: Yeast Colony PCR
Useful Websites
References
Index
Copyright
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This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.
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A catalog record for this book is available from the Library of Congress
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library
ISBN: 978-0-12-804231-1
For information on all Academic Press publications visit our website at https://www.elsevier.com/
Publisher: Sara Tenney
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Dedications
I dedicate this book to my father, Azad Najafov, who introduced me to the world of molecular biology and to my friend and mentor, İbrahim Barış, who taught me how to do my first PCR.
Ayaz Najafov
I dedicate this book to my family to whom I am eternally grateful for their constant support and care for me and my academic career.
Gerta Hoxhaj
Biography
Ayaz Najafov
Ayaz was born in Baku, Azerbaijan and as a high school student, participated in local and international biology Olympiads, where he had won several gold, silver, and bronze medals. He obtained his BSc and MSc degrees in Molecular Biology and Genetics from Bosphorus University, Istanbul, Turkey, and got a 2-year training in Biochemistry as an intern at Baylor College of Medicine, Houston, TX, United States. Ayaz pursued his PhD in Biochemistry at MRC Protein Phosphorylation Unit at Dundee University, Scotland, United Kingdom in Prof. Dario Alessi’s lab. Ayaz is currently a postdoctoral fellow in Prof. Junying Yuan’s lab at Harvard Medical School, Cell Biology Department.
Gerta Hoxhaj
Gerta was born in Fier, Albania. She obtained her BSc degree in Molecular Biology and Genetics, as well as a second major in Chemistry from Bosphorus University, Istanbul, Turkey. Gerta pursued her PhD in Biochemistry at MRC Protein Phosphorylation Unit at Dundee University, Scotland, United Kingdom in Prof. Carol MacKintosh’s lab. Gerta is currently a postdoctoral fellow in Prof. Brendan Mannings’s lab at Harvard T.H. Chan School of Public Health, Department of Genetics and Complex Diseases.
Preface
This book was written with an aim of providing researchers practicing molecular biology, whether at their beginner or expert stage, with a handy tool and reference for setting up, performing, optimizing, and troubleshooting polymerase chain reaction (PCR). We believe that this book will provide tremendous assistance for solving problems associated with challenging cases of PCR.
This book does not provide the reader with vast details of the PCR theory and the multiple applications of the technique. As this book is a laboratory guide, the emphasis here was made on the technical aspects of employing PCR as a tool in molecular biology laboratories and the issues were described in bench terms,
which makes the book more comprehendible to the end users of the technique.
General optimization and troubleshooting strategies, as well as a detailed