DRAFT
September 1, 2004 11:39 am, App_P&PDesign.fm
Assay Development Guidelines
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide B-3
• For multiplex PCR applications (involving multiple probes), design the probes
with different fluorescent reporter dyes (see Table B-1).
Design Primers for the Assay
Adhere to the following guidelines when designing primers for 5´-nuclease assays:
• Keep the G-C content in the range of 30–80%.
• Avoid runs of an identical nucleotide (especially guanine, where runs of four or
more bases should be avoided).
•Keep the T
m
in the range of 58-60 °C (using the Primer Express
™
software).
• Limit the G and/or C bases on the 3´ end. The five nucleotides at the 3´ end
should have no more than two G and/or C bases.
• Place the forward and reverse primers as close as possible to the probe without
overlapping the it.
• Use an annealing temperature of 58-60 °C for quantitative PCR, and 58-60 °C for
allelic discrimination (except for TaqMan
®
PDARs for Allelic Discrimination).
Quantitate the
Probes and
Primers
Use a spectrophotometric method to determine the concentrations of the probes and
primers received. See the TaqMan
®
Universal PCR Master Mix Protocol
(PN 4304449) or the TaqMan
®
Fast Universal PCR Master Mix (2
✕
) Protocol
(PN 4351891) for specific information about primer and probe quantification.
Prepare
Master Mix
Refer to the TaqMan
®
Universal PCR Master Mix Protocol (PN 4304449) or the
TaqMan
®
Fast Universal PCR Master Mix (2
✕
) Protocol (PN 4351891) for specific
information about preparing the master mix for use.
IMPORTANT! PCR master mix used with the 7900HT instrument must contain a
passive reference dye. The SDS software uses the signal from the passive reference
to normalize the reporter fluorescence making well-to-well comparisons possible.
All Applied Biosystems master mix products contain an optimal concentration of the
ROX passive reference dye.
Note: Applied Biosystems protocols are available on the Applied Biosystems
Company Web Site. See “How to Obtain Services and Support” on page xii for more
information.
Optimize
Primer/Probe
Concentrations
Refer to the TaqMan
®
Universal PCR Master Mix Protocol (PN 4304449) for
specific information about optimizing primer/probe concentrations.
Note: Applied Biosystems protocols are available on the Applied Biosystems
Company Web Site. See “How to Obtain Services and Support” on page xii for more
information.
Table B-1 Reporter Dyes for Multiplex PCR Applications
First Probe Second Probe
Reporter Dye
*
*The use of the FAM and VIC reporter dyes for multiplex applications provides the greatest
degree of spectral separation.
FAM
™
VIC
®
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