Real-Time PCR Troubleshooting

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Overview

Are you having trouble with your real-time PCR experiment? Do you need help diagnosing your experiment? The real-time PCR doctor is here to help. Unexpected fluorescence data are symptomatic of problems with your real-time PCR reaction components or amplification protocol. Click one of the symptoms below to learn about possible causes and treatments.

Related Topics: What Is Real-Time PCR?, How Real-Time PCR Works, and qPCR Assay Design and Optimization.

 

Gene Expression/Quantification Experiments:

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My experiment displays:

Poor Signal or No Signal
Poor signal or no signal
 		 Signal in Negative Control
Signal in negative control
 		 CT Values Differing Across Replicate Samples
Poor reproducibility across replicate samples		 Low or High Reaction Efficiency
Low or high reaction efficiency
 

Allelic Discrimination Experiments

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My experiment displays:

More than 3 clusters
More than 3 clusters		 Homozygotes as heterozygotes
Homozygotes as heterozygotes
 

High Resolution Melt (HRM) Experiments:

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My experiment displays:

Missed call or clustering
Missed call or clustering		 Amplification did not reach plateau
Amplification did not reach plateau		 Poor amplification or failed reaction
Poor amplification or failed reaction
 

If you don't see your symptom listed above, please contact technical support at 1-800-424-6723, option 2.

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Literature
Number Description Download
5692 Real-Time qPCR as a Tool for Evaluating RNAi-Mediated Gene Silencing, Rev A (tech note) Click to download
5962 Fast Optimization of a Multiplex Influenza Identification Panel Using a Thermal Gradient, Rev A Click to download
6004 A Practical Guide to High Resolution Melt Analysis Genotyping, Rev A Click to download
6009 High Resolution Melt Parameter Considerations for Optimal Data Resolution, Rev A Click to download
6047 Optical Design of CFX96 Real-Time PCR Detection System Eliminates the Requirement of a Passive Reference Dye, Rev A Click to download
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