Adding Custom Dyes To The Pure Dye Set - Applied Biosystems 7900HT User Manual

Adding Custom Dyes to the Pure Dye Set

When to Perform
Materials
Required
Creating a
Custom Pure Dye
Plate
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
The Applied Biosystems 7900HT Fast Real-Time PCR System can be used to run
assays designed with custom dyes (dyes not manufactured by Applied Biosystems).
However, before using custom dyes with the 7900HT instrument, you must create
and run a custom pure dye plate.
• ABI P ™ 384- or 96-Well Optical Reaction Plate or Optical 96-Well Fast
RISM
Thermal Cycling Plate
• An optical adhesive cover or ABI P
• Custom Dye(s)
• Pipettor, 100-µL (with pipet tips)
• Centrifuge, with plate adaptor
The purpose of the custom pure dye plate is identical to that of an ABI P
Note:
Pure Dye Plate. The SDS software uses the custom plate to create a spectral standard
for multicomponenting the custom dye.
1. Prepare a microplate with a dilution series of the custom dye.
2. Start the SDS software.
3. Create an allelic discrimination plate document and run the dilution series plate.
It is not necessary to configure detector, sample, and method information for
Note:
the dilution series 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.
4. Select Analysis > Analyze.
The software analyzes the raw run data.
5. Click (Show Raw Data Plot).
6. In the Raw Data Plot, determine the highest concentration of dye that does not
produce a saturated signal, and record it for future use.
Saturated signals are characterized by their high peaks that rise beyond
Note:
detectable levels (> 65,000 fluorescent units) and appear as plateaus on the
Raw Data plot.
The concentration of the custom dye that yields the highest possible signal but
does not saturate is the maximum concentration for use with the 7900HT
instrument.
7. Repeat steps 1 through 6

Adding Custom Dyes to the Pure Dye Set

™ Optical Flat Caps
RISM
for any additional custom dyes.
DRAFT
September 1, 2004 11:39 am, CH_Maintenance.fm
RISM
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