ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Real-Time Runs (Quantitative PCR and Dissociation Curves)
Troubleshooting
Analyzed Data from
aReal-TimeRun
When faced with irregular data, you can use the SDS software to diagnose some
chemistry- and instrument-related problems. The following table contains a summary
of checks for verifying the integrity of your run data and to help you begin
troubleshooting potential problems.
Troubleshooting Analyzed Real-Time Run Data
Analysis View/Description What to look for...
Raw Data Plot
Signal tightness and uniformity – Do the raw spectra signals
from replicate groups and controls exhibit similar spectral
Displays the raw reporter
‘profiles’? If not, the plate or sample block could be
fluorescence signal
contaminated.
(not normalized) for the
selected wells during each Characteristic signal shape – Do the samples peak at the
cycle of the real-time PCR. expected wavelengths? For example, samples containing
only FAM- labeled TaqMan® probes should not produce
raw fluorescence in the wavelength of a VIC dye
component. A signal present in wells that do not contain the
dye could indicate that the sample, master mix, or well
contains contaminants.
Characteristic signal growth – As you drag the bar through
the PCR cycles, do you observe growth as expected?
Absent growth curves may indicate a pipetting error (well
lacks template).
Signal Plateaus – Do any of the signals plateau? Signal
plateaus or saturation can be an indication that a well
contains too much template or fluorescent signal.
Multicomponent Plot
Correct dyes displayed – Does the plot display all dyes as
expected? The presence of an unexpected dye may be the
Displays a plot of normalized
result of an error in detector setup, such as assigning the
multicomponent data from a
wrong reporter or quencher dye.
single well of a real-time run.
The plot displays the ROX fluorescence level – Does the ROX signal fluoresce
component dye signals that below the reporter dyes? If not, the lack of reporter
contribute to the composite fluorescence may be caused by an absence of probe in the
signal for the well.
well (a pipetting error).
Background fluorescence – Do all dyes fluoresce above the
background? The Background signal is a measure of
ambient fluorescence. If a dye fails to fluoresce above the
background, it is a strong indication that the well is missing
probes labeled with the dye (well does not contain probe,
PCRmastermix,orboth).
MSE Level – The MSE (mean squared error) is a
mathematical representation of how accurately the
multicomponented data fits the raw data. The higher the
MSE value, the greater the deviation between the
multicomponented data and the raw data.
Troubleshooting 8-11