Applied Biosystems 7900HT User Manual

DRAFT
September 1, 2004 11:39 am, CH_Trouble.fm
End-Point Runs (Allelic Discrimination)
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 8-13
Amplification Plot
The Amplification plot displays data from real-time runs after signal normalization
and Multicomponent analysis. It contains the tools for setting the baseline and
threshold cycle (C
T
) values for the run.
What to look for:
• Correct baseline and threshold settings – Are the baseline and threshold
values set correctly?
Identify the components of the amplification curve and set the baseline so that
the amplification curve growth begins at a cycle number that is greater than the
highest baseline number.
IMPORTANT! Do not adjust the default baseline if the amplification curve
growth begins after cycle 15.
Identify the components of the amplification curve and set the threshold so that it is:
• Above the background
• Below the plateaued and linear regions
• Within in the geometric phase of the amplification curve
• Irregular amplification – Do all samples appear to have amplified normally? The
three phases of the amplification curve should be clearly visible in each signal.
• Outlying amplification – When the run data is viewed in the C
T
vs. Well
Position plot, do replicate wells amplify comparably? Wells producing C
T
values that differ significantly from the average for the associated replicate
wells may be considered outliers.
If a plate produces non-uniformity between replicates, some samples on the plate
could have evaporated. Check the seal of the optical adhesive cover for leaks.
End-Point Runs (Allelic Discrimination)
Troubleshooting
Analyzed Data
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.
Raw Data
The Raw Data Plot displays the raw reporter fluorescence signal (not normalized) for
the selected wells during each cycle of the PCR.
What to look for...
• Signal tightness and uniformity – Do the raw spectra signals from replicate
groups and controls exhibit similar spectral ‘profiles’? If not, the plate or
sample block could be contaminated.
• Characteristic signal shape – Do the samples peak at the expected
wavelengths? For example, samples containing only FAM dye-labeled TaqMan
probes should not produce raw fluorescence in the peak wavelength of the 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.
• 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.

Applied Biosystems 7900HT Specifications

General

Brand	Applied Biosystems
Model	7900HT
Category	Laboratory Equipment
Language	English