Bio-Rad iQ™5 Optical System Software, Version 2.1 - Manual (Page 52)

Section 5 Run-Time Central Module
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Persistent well factors are stored in the Well Factors folder in the iQ5 Folder. Refer to section 7
for information on the calibration and generation of persistent well factors.
In general, persistent well factors can be used for approximately three months, but should be re-
generated anytime something pertinent to the optical system is changed such as the optical
filters or the camera lamp. A warning reminder to re-calibrate to generate new persistent well
factors will occur when Persistent Well Factors are older than 90 days.
5.3 Real-Time PCR Experiments Using DNA-Binding Dyes
In most real-time PCR experiments using DNA-binding dyes, like SYBR ® Green I or ethidium
bromide, dynamic well factors may not be collected. When the template DNA is denatured, the
fluorescence of these dyes is not sufficiently high to calculate statistically valid well factors using
the experimental plate.
There are three possible solutions to this problem:
•
Use Bio-Rad iQ™ SYBR ® Green supermix which contains fluorescein, enabling dynamic
well factors to be collected directly from the experimental plate
•
Use persistent well factors
•
Spike reaction mixes that do not come premixed with fluorescein with fluorescein to a
final concentration of 10 nM. The addition of fluorescein provides sufficient fluorescence
at 95°C for the collection of well factors from the experimental plate while not interfering
with the PCR reaction
5.3.1 Spiking Real-Time PCR Experiments Using DNA binding Dyes
With Fluorescein
The iQ SYBR ® Green supermix is already spiked with a small amount of fluorescein that permits
the collection of well factors from the experimental plate. It is also possible to collect well factors
from the experimental plate with other commercial SYBR
® Green mixes or with home-brew mixes
by adding sufficient fluorescein to bring the reaction mixture to 10 nM fluorescein.
Prepare a 1 mM solution of fluorescein by making a 1:1000 dilution of the 1 mM stock fluorescein
calibration dye in PCR buffer (10 mM Tris, pH 8.0, 50 mM KCl, 3 mM MgCl
2
). Then mix 1 part of
the 1 mM fluorescein with 990 µl of master mix to yield a final concentration of 10 nM
fluorescein. Once well factors are collected from the experimental plate, they are written to the
.opd file and the software continues to execute the programmed protocol.
5.4 Initiate Run Window: Run End Point Selected
Initiate an End Point run by selecting a Plate Setup, then click Run End Point. The software
transfers to the Initiate Run window in the Run-Time Central module. An End Point run uses a
predefined canned protocol in which only the temperature of the data collection step can be
modified. Enter the desired temperature of the data collection step into the Setpoint box as
shown in Figure 5.3. Click Enter. Changes entered into the Setpoint box appear in the Selected
Protocol region of the Initiate Run window.