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Sigmoidal amplification curves
If your amplification curves look sigmoidal, it is likely you have one of these problems:
If you set the end cycle of the baseline setting too low, insufficient background signal will be subtracted from some or all samples. As a result, the precision of affected curves will be lowered. In addition, amplification curves for affected samples may assume a sigmoidal shape.
If you have selected Manual Baseline to analyze your data, the upper baseline setting must be set to accommodate the sample with the earliest CT. In this instance, samples whose amplification curves appear later in the run may not have sufficient background subtracted.
The default Manual Baseline End Cycle setting on all Applied Biosystems real-time PCR instruments is 15. If a run contains samples whose amplification curves significantly appear after cycle 15 and you neglect to adjust the upper baseline setting, there will be an insufficient amount of background subtraction.
Analyzing with the Auto Baseline setting typically solves the problem in either of the previous two instances, since the Auto feature subtracts the optimal background signal on a sample-by-sample basis.
For more information about how to properly analyze your data, including Auto or Manual Baseline settings, please refer to the Data Analysis section of your instrument Getting Started Guides. These documents are available on our site's Troubleshooting & Tutorials page.
If one or more samples on a plate contains an unusually high level of fluorescent noise during the early cycles of PCR, the amplification curves of these samples may take on an irregular (typically sigmoidal) shape. Though rare, this effect can be seen when using the Auto Baseline analysis feature.
High fluorescent noise may be caused by one of two factors:
For Research Use Only. Not for use in diagnostic procedures.