Your amplification curves have a sigmoidal shape
Sigmoidal amplification curves
If your amplification curves look sigmoidal, it is likely you have one of these problems:
The baseline setting in your instrument data analysis software may be too low
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.
You may have a high level of fluorescent noise during the early cycles of PCR
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:
The assay itself may have an unusual amount of background signal, in which case all samples for a particular assay are likely to take on an irregular shape:
This happens more commonly with SYBR® Green than with TaqMan® assays, and is often corrected by switching chemistries. Otherwise, to correct for the problem during data analysis, you may try switching to Manual Baseline for the offending assay.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 Tutorials & Troubleshooting page.
One or more samples were not adequately mixed prior to cycling:
In these cases, only a few samples for a given assay may have an irregular shape. Inadequate mixing is generally caused by failing to mix the aqueous sample fully with the master mix (which is somewhat viscous due to its glycerol content). This effect, in turn, is most often seen when one of two scenarios occurs:
- The volume of sample exceeds 20% of the final reaction volume—the larger the sample volume is with respect to the master mix/cocktail, the more difficult it becomes for these two components to mix adequately.
- Applied Biosystems Fast Master Mix is being used, and you have failed to centrifuge your plate prior to cycling—because the fast master mix requires only a twenty-second hot-start at 95°C (compared to the ten-minute hot-start employed by standard master mixes), centrifuging prior to cycling is especially critical.