Contents
iii
1 Introduction
Overview 1-1
About This Chapter. 1-1
In This Chapter 1-1
Purpose of the Kit . 1-2
About the Kit . 1-2
Basics of the 5´ Nuclease Assay . 1-2
TaqMan Probe. 1-5
AmpliTaq Gold DNA Polymerase . 1-5
TaqMan Universal PCR Master Mix1-5
Materials and Equipment1-6
Kit Components . 1-6
Materials Required but Not Supplied . 1-6
Storage and Stability 1-8
Safety . 1-9
Documentation User Attention Words 1-9
Chemical Hazard Warning . 1-9
Chemical Waste Hazard Warning . 1-10
Site Preparation and Safety Guide 1-10
About MSDSs 1-10
Ordering MSDSs1-11
Preventing Contamination 1-12
Overview1-12
False Positives1-12
AmpErase UNG Inactivation 1-13
Prevention of PCR Product Carryover . 1-13
General PCR Practices . 1-13
Fluorescent Contaminants 1-14
2 Amplifying Custom Target Sequences for Quantitation
Overview2-1
About This Chapter 2-1
In This Chapter2-1
Identifying Target Sequence and Amplicon Size . 2-2
Target Template Defined 2-2
Amplicon Defined . 2-2
Amplicon Size 2-2
Designing TaqMan Probes and Primers 2-3
Probes. 2-3
Primers 2-3
Quantitating Probes and Primers . 2-4
Method 2-4
Optimizing Primer and Probe Concentrations . 2-5
Determining Optimal Primer Concentration . 2-5
PCR Reaction Mix for Primer Optimization . 2-6
Thermal Cycling Conditions for Primer Optimization . 2-7
Determining Optimal Probe Concentration 2-7
Procedure 2-7
Reaction Mix for Probe Optimization 2-8
Thermal Cycling Conditions for Probe Optimization 2-8
Performing Routine Analysis 2-9
Overview 2-9
3 Amplifying Custom Sequences for Allelic
Discrimination
Overview3-1
About This Chapter 3-1
In This Chapter3-1
Identifying Target Sequence and Amplicon Size . 3-2
Target Template Defined. 3-2
Amplicon Size. 3-2
Designing the TaqMan Probe and Primers 3-3
Probes . 3-3
Primers 3-3
Quantitating Probes and Primers 3-4
Method 3-4
Optimizing Probe and Primer Concentrations3-5
Probe Concentrations . 3-5
Determining Optimal Probe Concentrations3-5
Default Assay Conditions 3-5
Determining Optimal Primer Concentrations . 3-5
4 Reverse
Transcription
Overview 4-1
About This Chapter. 4-1
In This Chapter 4-1
Reverse Transcription for All Amplicons Except 18S . 4-2
Overview. 4-2
Guidelines 4-2
Choice of Primers 4-2
Performing RT Reactions 4-3
Thermal Cycling . 4-5
Reverse Transcription for the 18S Amplicon 4-6
Overview. 4-6
Recommended Template. 4-6
Template Quality. 4-6
Template Quantity4-6
Guidelines 4-7
Preparing the Reactions . 4-8
Thermal Cycling 4-10
5 Data Analysis
Overview5-1
About This Chapter 5-1
In This Chapter5-1
Interpreting the Results . 5-2
Normalization. 5-2
Multicomponenting 5-2
Rn and ΔRnValues5-2
Real-Time Detection . 5-4
Threshold Cycle . 5-4
A Troubleshooting
B References
C Technical Support
Services & Support C-1
Applied Biosystems Web Site C-1