IDT offers several free, online tools for qPCR probe design: the PrimeTime qPCR
, RealTime PCR
, and PrimerQuest
tools. Because these tools contain design engines that use sophisticated formulas 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. Contact IDT Technical Support (firstname.lastname@example.org
) if you require help with custom designs.