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Real-Time PCR Troubleshooting Tool

Having problems with your gene expression or SNP genotyping experiments? Do your amplification curves look sigmoidal, or do you have no curves at all? Do your allelic discrimination plots have diffuse or trailing clusters? Our interactive troubleshooting tool will guide you step by step to a solution.


Gene Expression/Quantitation Experiments

I am having problems with...


Abnormal Amplification Abnormal Amplification
Your curves are sigmoidal or amplification occurs later than you expected.
NTC Positive Amplification NTC Positive Amplification
You have an amplification product in your no template control (NTC).
Abnormal Amplification No Amplification
You have no detectable PCR product.
Abnormal Amplification Poor PCR Efficiency
If the slope of your standard curve is below
-3.6, you have poor PCR efficiency.


Example of a normal amplification curve


Abnormal Amplification Normal Amplification Curve
Your amplification curve should look something like this.



 
SNP Genotyping Experiments

I am having problems with...


SNP Not Amplifying SNP Not Amplifying
You cannot amplify a SNP.
Trailing Custers Trailing Clusters
Not using ROX or not designating it as a passive reference dye may cause trailing clusters.
Diffuse Clusters Diffuse Clusters
You have diffuse clustering in your SNP analysis.
No Autocalling No Autocalling
Failure of autocalling of SNP results is usually due to outlier wells or software problems.


Example of a normal allelic discrimination plot


Normal Allelic Discrimination Plot Normal Allelic Discrimination Plot
Your cluster plot should look something like this.



     
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