Introduction to Quantitative PCR: Methods and Application Guide

Stratagen real-time PCR techbook. It is downloadable as a 50 pages pdf file.

Table of Contents:

Real-Time vs. Endpoint Quantitative PCR ...................................................................................................................................................3
Experimental Design ........................................................................................................................................................................................5
QPCR Chemistry Options ................................................................................................................................................................ 6
Methods of Quantification.............................................................................................................................................................. 9
Controls.............................................................................................................................................................................................. 12
Primer and Probe Design................................................................................................................................................................14
Dye and Quencher Choice...............................................................................................................................................................16
Probe and Primer Synthesis..........................................................................................................................................................................18
QRT-PCR Reactions: One-step vs. Two-step ...............................................................................................................................................19
Reagent Choice ............................................................................................................................................................................................... 20
Reference Dye Considerations ..................................................................................................................................................... 20
QPCR Reagents................................................................................................................................................................................ 20
Assay Optimization.........................................................................................................................................................................................23
Primer Optimization Guidelines ..................................................................................................................................................23
Probe Concentration Optimization Guidelines ..........................................................................................................................25
Standard Curves.............................................................................................................................................................................. 26
Further Optimization......................................................................................................................................................................27
Multiplex Assay Considerations...................................................................................................................................................27
The Ideal Assay................................................................................................................................................................................ 28
QPCR Experiment Data Analysis ................................................................................................................................................................ 29
Ensuring Your Ct Values Are Accurate....................................................................................................................................... 29
Standard Curve Quantification.................................................................................................................................................... 34
Relative or Comparative Quantification.....................................................................................................................................35
Qualitative PCR ............................................................................................................................................................................... 36
References........................................................................................................................................................................................................ 38
Endnotes........................................................................................................................................................................................................... 38
Appendix .......................................................................................................................................................................................................... 39
Plate Set-up Example ..................................................................................................................................................................... 39
Probe Optimization Reaction Example......................................................................................................................................40
QPCR Glossary..................................................................................................................................................................................41
QPCR References & Useful Websites .......................................................................................................................................... 45
Reagent & Ordering Information.................................................................................................................................................46

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