Table of Contents

Preface
Chapter 1: Introduction
      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
      2.1. Primer Design
      2.2. Standard PCR setup
      2.3. Controls
      2.4. Cycling Parameters
Chapter 3: Good PCR Practices
Chapter 4: Troubleshooting PCR Problems
      4.1 Optimization rules
      4.2 General Optimization Strategies
          4.2.1. Annealing temperature gradient
          4.2.2. Mg++ Titration
          4.2.3. Hot-start
          4.2.4. Touchdown
          4.2.5. Additives
      4.3 Troubleshooting Specific Problems
          4.3.1. Problem type 1: No PCR products were observed.
          4.3.2. Problem type 2: Multiple PCR products were observed.
          4.3.3. Problem type 3: Smears were observed.
          4.3.4. Problem type 4: Observed bands were faint (low yield).
          4.3.5. Problem type 5: Bands observed in controls.
          4.3.6. Other Problem Types.
Chapter 5: Practical Tips
Chapter 6: Special Cases
      6.1. Long-PCR
      6.2. RAPD-PCR
      6.3. Multiplex PCR
Appendix A: Agarose Gel Electrophoresis Protocol
Appendix B: Silver Staining of DNA Gels
Appendix C: Plasmid Miniprep Protocol
Appendix D: Restriction Enzyme Digestion Protocol
Appendix E: Protocol for DNA Concentration Estimation by using Hoechst 33258
Appendix F: Formulas
Appendix G: Primers for Common Promoters, Housekeeping Genes, 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
Glossary and Abbreviations
Index