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How can I design primers and probes for qPCR?
IDT offers several free, online tools for qPCR primer and probe design: PrimeTime Predesigned qPCR Assays design tool, PrimerQuest Tool, OligoAnalyzer Tool, and RealTime PCR Tool guide you through identifying primer and probe sequences to making sure they are specific to your assay. Because these tools contain design engines that use sophisticated algorithms that, for example, take into account nearest neighbor analysis to calculate Tm, they generally provide the very best designs.
Keep in mind: Probes used in qPCR should have Tm at least 6 degrees higher than the flanking primers, and should be 28 bases or shorter. Probe designs should be screened for self-dimers, hetero-dimers against the primers, as well as hairpins. Also, run a BLAST alignment to ensure probe efficiency will not be reduced due to off target interactions. For more information review IDT primer design tools for PCR & qPCR or contact IDT technical support (email@example.com) for help with custom designs.