Applied Biosystems 7900HT User Manual page 335

Quantitate the
Probes and
Primers
Prepare
Master Mix
Optimize
Primer/Probe
Concentrations
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
• For multiplex PCR applications (involving multiple probes), design the probes
with different fluorescent reporter dyes (see
Table B-1 Reporter Dyes for Multiplex PCR Applications
Reporter Dye
*
*The use of the FAM and VIC reporter dyes for multiplex applications provides the greatest
degree of spectral separation.
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 in the range of 58-60 °C (using the Primer Express
m
• 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
Use a spectrophotometric method to determine the concentrations of the probes and
primers received. See the TaqMan
(PN 4304449) or the TaqMan
(PN 4351891) for specific information about primer and probe quantification.
Refer to the TaqMan ® Universal PCR Master Mix Protocol (PN 4304449) or the
TaqMan ® Fast Universal PCR Master Mix (2
information about preparing the master mix for use.
PCR master mix used with the 7900HT instrument must contain a
IMPORTANT!
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.
Applied Biosystems protocols are available on the Applied Biosystems
Note:
Company Web Site. See
information.
Refer to the TaqMan ® Universal PCR Master Mix Protocol (PN 4304449) for
specific information about optimizing primer/probe concentrations.
Applied Biosystems protocols are available on the Applied Biosystems
Note:
Company Web Site. See
information.
First Probe
FAM ™
® PDARs for Allelic Discrimination).
® Universal PCR Master Mix Protocol
® Fast Universal PCR Master Mix (2
✕
) Protocol (PN 4351891) for specific
"How to Obtain Services and Support"
"How to Obtain Services and Support"
DRAFT
September 1, 2004 11:39 am, App_P&PDesign.fm
Assay Development Guidelines
Table B-1).
Second Probe
VIC ®
™ software).
✕
) Protocol
on page xii
on page xii
for more
for more
B-3