Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Assay Development Guidelines• For multiplex PCR applications (involving multiple probes), design the probeswith different fluorescent reporter dyes (see Table B-1).Table B-1Reporter Dyes for Multiplex PCR ApplicationsFirst ProbeSecond ProbeReporter Dye *FAM VIC ®*The use of the FAM and VIC reporter dyes for multiplex applications provides the greatestdegree of spectral separation.Design Primers for the AssayAdhere 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 ormore 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´ endshould have no more than two G and/or C bases.• Place the forward and reverse primers as close as possible to the probe withoutoverlapping the it.• Use an annealing temperature of 58-60 °C for quantitative PCR, and 58-60 °C forallelic discrimination (except for TaqMan ® PDARs for Allelic Discrimination).Quantitate theProbes andPrimersPrepareMaster MixOptimizePrimer/ProbeConcentrationsUse a spectrophotometric method to determine the concentrations of the probes andprimers 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.Refer to the TaqMan ® Universal PCR Master Mix Protocol (PN 4304449) or theTaqMan ® Fast Universal PCR Master Mix (2✕) Protocol (PN 4351891) for specificinformation about preparing the master mix for use.IMPORTANT! PCR master mix used with the 7900HT instrument must contain apassive reference dye. The SDS software uses the signal from the passive referenceto normalize the reporter fluorescence making well-to-well comparisons possible.All Applied Biosystems master mix products contain an optimal concentration of theROX passive reference dye.Note: Applied Biosystems protocols are available on the Applied BiosystemsCompany Web Site. See “How to Obtain Services and Support” on page xii for moreinformation.Refer to the TaqMan ® Universal PCR Master Mix Protocol (PN 4304449) forspecific information about optimizing primer/probe concentrations.Note: Applied Biosystems protocols are available on the Applied BiosystemsCompany Web Site. See “How to Obtain Services and Support” on page xii for moreinformation.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide B-3DRAFTSeptember 1, 2004 11:39 am, App_P&PDesign.fm