Chapter 6
Performing the Pure Dye Calibration
Overview
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation and Maintenance Guide
69
Notes
6
Purpose of Pure
Dye Calibration
During a pure dye calibration run, the system:
• 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.
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.
Dye Sets
The Applied Biosystems Real-Time PCR Systems use the following dye sets for
calibration:
•
7300 system
– FAM
™
, JOE
™
, NED
™
, ROX
™
, TAMRA
™
, VIC
®
, and the SYBR
®
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).
The following figure shows the emission spectrum for each dye, and the filters and
wavelengths at which each dye is read.
600
500
700
Filters
SDS
System
7300 System
7500/7500 Fast System
Spectrum
(nm)
Emission
Spectra
Pure Dyes
A
B
C
D
E
~520 nm
FAM
SYBR Green
~550 nm
VIC
~580 nm
TAMRA
~610 nm
~670 nm
JOE
NED
CY3
CY5
TEXAS RED
ROX
Summary of Contents for 7300
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