Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 51)

Section 5 Run-Time Central Module
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Fig. 5.2. The Initiate Run Window.
5.2 Well Factors
Well Factors are used to compensate for any system or pipeting non-uniformity in order to
optimize fluorescent data quality and analysis. Well Factors may be collected directly from the
experimental plate (dynamic well factors) or from an external well factor plate (persistent well
factors).
The best source of well factors, for correcting non-uniformity, are dynamic well factors collected
from the actual experimental plate. However, in order to collect well factors from the
experimental plate, the plate must meet certain requirements and be cycled for approximately 5
extra minutes.
5.2.1 Dynamic Well Factors
In order to collect dynamic well factors one of the following must be true:
• All wells must have the same combination of fluorophores AND the same concentration
of fluorophores
• No fluorophore is used both alone and in combination with other fluorophores AND no
fluorophore is used in more than one combination of fluorophores
The collection of dynamic well factors is a completely automated process, which begins as soon
as Begin Run is clicked and the file name saved. When collecting dynamic well factors, the plate
is held at 95°C for 2.5 minutes prior to the first cycle with a setpoint of 90.0°C or higher.
5.2.2 Persistent Well Factors
When setting up a plate setup to run the experiment specific reaction vessel and sealing methods
are chosen. This plate setup will not be able to be used to run an experiment unless persistent
well factor and background calibration files with matching vessel and sealing type have already
been collected.