Applied Biosystems 7300 Installation And Maintenance Manual page 95

During a pure dye calibration run, the system:
Purpose of Pure
Dye Calibration
The software then uses the pure spectra data during subsequent runs to characterize pure
dyes and distinguish the individual contribution of each dye in the collective
fluorescence collected by the instrument during a run.
After each run, the SDS software receives run data in the form of a raw spectra signal for
each reading. It determines the contribution of each fluorescent dye used in the sample
by comparing the raw spectra to the pure spectra file. When you save a plate document
after analysis, the software stores the pure spectra file with the collected fluorescence
data for that experiment.
The Applied Biosystems Real-Time PCR Systems use the following dye sets for
Dye Sets
calibration:
The following figure shows the emission spectrum for each dye, and the filters and
wavelengths at which each dye is read.
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation and Maintenance Guide
• Collects spectral data from a series of pure dye standards.
• Stores the spectral information for the pure dye standards in the pure spectra run
file, a calibration file in the SDS directory.
™
• 7300 system – FAM , JOE
Green I dsDNA binding dye, inside preloaded 96-well pure dye plates.
• 7500 and 7500 Fast systems – All of the dyes listed above, plus CY3, CY5, and
®
TEXAS RED dyes (because of the additional filter).
SDS
System
Filters
Spectrum
(nm)
Emission
Spectra
Pure Dyes ~520 nm
FAM
SYBR Green
Chapter 6 Performing the Pure Dye Calibration
™ ™ ™
, NED , ROX , TAMRA
7500/7500 Fast System
7300 System
A B C D
500 600
~550 nm ~580 nm
JOE TAMRA
VIC NED
CY3
Overview
™ ®
, VIC , and the SYBR
E
700
~610 nm ~670 nm
ROX
CY5
TEXAS RED
®
6
69