Amplification plot
The Amplification Plot screen displays post-run amplification of the samples of each experiment added to your project. Three plots are available:
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ΔRn vs Cycle—ΔRn is the magnitude of normalized fluorescence signal generated by the reporter at each cycle during the PCR amplification (ΔRn = Rn – baseline). This plot displays ΔRn as a function of cycle number. You can use this plot to identify and examine irregular amplification and to view threshold and baseline values for the run.
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Rn vs Cycle—Rn is the fluorescence signal from the reporter dye that is normalized to the fluorescence signal from the passive reference. This plot displays Rn as a function of cycle number. You can use this plot to identify and examine irregular amplification.
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CT vs Well—CT (Cq) is the PCR cycle number at which the fluorescence meets the threshold in the amplification plot. This plot displays CT as a function of well position. You can use this plot to find outlying amplification (outliers).
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Toolbar—Contains the following tools for controlling the plot:
—Select individual data points from the plot.
—Zoom the plot to the selected area.
—Zooms out the plot to show all data points.
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Threshold—The threshold (calculated or manual) that is currently applied to the project data.
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View Options—The view options for the Amplification Plot. Use the dropdown lists to select the plot displayed by the software (ΔRn vs Cycle, Rn vs Cycle, or CT vs Well), the scale of the y-axis (log or linear), and the color scheme for the plot.
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Amplification curves—Normalized fluorescence for individual wells throughout the course of the thermal cycling protocol.
—Saves the plot as an image (PNG or JPG).
—Allows you to adjust the view options for the plot.
Note: When the Amplification Plot displays a large data set, the Applied Biosystems™ Software alters the appearance of curves for better identification:
View large data sets
When an Amplification Plot for a sample, target, or plate includes data for more than 384 data points, the Applied Biosystems™ Software displays a subset of the data (shown in color) for active viewing against a background of the full data set (shown in gray). The range of active data in the Amplification Plot is controlled through the histogram beneath the plot, which displays the well data for the experiment tabulated by an associated numerical attribute (such as Amp Score or CT/CRT without Flags).
The position of the anchor icon (
) in the histogram controls the range of active data displayed by the Amplification Plot, where the software displays the curves from the 384 data points with values nearest to the position of the icon. To change the range of active data, click and drag the icon to view the data nearest the desired location.
Note: Alternatively, click
/
below the Well Table to advance the active data range displayed by the Amplification Plot.
The table heading that is selected in the Well Table determines the content of the histogram. By default, the histogram tabulates data by Amp Score as shown above. To change the data that are displayed on the x-axis of the histogram, select the heading of a column in the Well Table that contains numerical content (such as, Amp Score or CT/CRT without Flags).
Note: Selecting the heading of a column in the Well Table that contains less than 384 data points hides the histogram. The feature is present only when the plot contains more than 384 amplification curves.