Low Precision Or Irreproducibility - Applied Biosystems 7900HT User Manual

Low Precision or Irreproducibility

Overview
Improper
Threshold Setting
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
There are many reasons why an assay run with the Applied Biosystems 7900HT Fast
Real-Time PCR System can have less than optimal precision. Factors that can affect
precision are described in detail below.
Improper Threshold Setting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Imprecise Pipetting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Non-Optimized Chemistry
Incomplete Mixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Air Bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Splashing PCR Reagents
Drops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Writing on the Reaction Plates
Fluorescent Contamination on the Plates
Errors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contaminated Sample Block
Improper or Damaged Plastics
Low Copy Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Use of Non-Applied Biosystems PCR
The key to high-precision quantitative PCR is accurate detection of the geometric
phase. The Applied Biosystems 7900HT Fast Real-Time PCR System typically
delivers sufficient sensitivity so that at least three cycles of the geometric phase are
visible, assuming reasonably optimized PCR conditions. The SDS software
calculates a fixed signal intensity, called a threshold, that each signal generated from
PCR amplification must reach before it is recognized by the software as actual
amplification. The calculated threshold is an approximation, and should be examined
and modified as needed.
Modifying the Threshold
In a real-time document of the SDS software, the threshold can be modified via the
Amplification plot view following analysis of the run data. See
and Threshold Values" on page 6-46
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Reagents. . . . . . . . . . . . . . . . . . . . . . . . . .
for more information.
DRAFT
September 1, 2004 11:39 am, CH_Trouble.fm
Low Precision or Irreproducibility
"Setting the Baseline
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