【分享】Molecular Biology Problem Solver: A Laboratory Guide.
丁香园论坛
1066
PCR问题及解决
内容：
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Developing a PCR Strategy: The Project Stage . . . . . . . . . . . . . 293
Assess Your Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Identify Any Weak Links in Your PCR Strategy . . . . . . . . . . 295
Manipulate the Reaction to Meet Your Needs . . . . . . . . . . 296
Developing a PCR Strategy: The Experimental Stage . . . . . . . 296
What Are the Practical Criteria for Evaluating a DNA
Polymerase for Use in PCR? . . . . . . . . . . . . . . . . . . . . . . . . 296
How Can Nucleotides and Primers Affect a PCR
Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
How Do the Components of a Typical PCR Reaction
Buffer Affect the Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . 305
How Can You Minimize the Frequency of Template
Contamination? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
What Makes for Good Positive and Negative
Amplification Controls? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
What Makes for A Reliable Control for Gene
Expression? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
How Do the Different Cycling Parameters Affect a
PCR Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Instrumentation: By What Criteria Could You
Evaluate a Thermocycler? . . . . . . . . . . . . . . . . . . . . . . . . . . 309
How Can Sample Preparation Affect Your Results? . . . . . . . 311
How Can You Distinguish between an Inhibitor
Carried over with the Template and Modification of
the DNA Template? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
What Are the Steps to Good Primer Design? . . . . . . . . . . 312
Which Detection and Analysis Strategy Best Meets
Your Needs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
Appendix A: Preparation of Plasmid DNA for Use as PCR
Controls in Multiple Experiments . . . . . . . . . . . . . . . . . . . . . . . . 327
Appendix B: Computer Software for Selecting
Primers . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Appendix C: BLAST Searches . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
Appendix D: Useful Web Sites . . . . . . . . . . . . . . . . . . . . . . . . . 328
内容：
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Developing a PCR Strategy: The Project Stage . . . . . . . . . . . . . 293
Assess Your Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Identify Any Weak Links in Your PCR Strategy . . . . . . . . . . 295
Manipulate the Reaction to Meet Your Needs . . . . . . . . . . 296
Developing a PCR Strategy: The Experimental Stage . . . . . . . 296
What Are the Practical Criteria for Evaluating a DNA
Polymerase for Use in PCR? . . . . . . . . . . . . . . . . . . . . . . . . 296
How Can Nucleotides and Primers Affect a PCR
Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
How Do the Components of a Typical PCR Reaction
Buffer Affect the Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . 305
How Can You Minimize the Frequency of Template
Contamination? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
What Makes for Good Positive and Negative
Amplification Controls? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
What Makes for A Reliable Control for Gene
Expression? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
How Do the Different Cycling Parameters Affect a
PCR Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Instrumentation: By What Criteria Could You
Evaluate a Thermocycler? . . . . . . . . . . . . . . . . . . . . . . . . . . 309
How Can Sample Preparation Affect Your Results? . . . . . . . 311
How Can You Distinguish between an Inhibitor
Carried over with the Template and Modification of
the DNA Template? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
What Are the Steps to Good Primer Design? . . . . . . . . . . 312
Which Detection and Analysis Strategy Best Meets
Your Needs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
Appendix A: Preparation of Plasmid DNA for Use as PCR
Controls in Multiple Experiments . . . . . . . . . . . . . . . . . . . . . . . . 327
Appendix B: Computer Software for Selecting
Primers . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Appendix C: BLAST Searches . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
Appendix D: Useful Web Sites . . . . . . . . . . . . . . . . . . . . . . . . . 328
