Appendix C
Determining Optimum Dye Concentration
128
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation and Maintenance Guide
Notes
Determining Optimum Dye Concentration
IMPORTANT!
Wear powder-free
gloves while creating a custom pure
dye plate.
1.
In the center wells of a 96-well plate, prepare a
dilution series of the custom dye (for example,
25, 50, 100, 200, 400, 800, 1600, and 3200 nM
concentrations) using 50-
µ
L volumes for the
7300/7500 system or 20-
µ
L volumes for the
7500 Fast system.
2.
Seal the wells of the reaction plate using an
optical adhesive cover.
3.
In the SDS software, click
(or
select
File New
).
4.
In the New Document dialog box, click
.
Note:
It is not necessary to configure detector,
sample, and method information for the plate
document. The purpose of the run is to establish
the correct working concentration for the dye by
viewing the intensity of the raw spectra produced
by the wells in the dilution series.
5.
Load the prepared plate:
a.
Press the tray door to open it.
b.
Load the pure dye plate into the plate
holder.
c.
Press the tray door to move the drawer into
the instrument.
25
50 100 200
1600 3200
400 800
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