FAQs

Yes. Clara® Inhibitor-Tolerant Probe 1-Step Mixes support ultra-fast cycling RT-qPCR, with successful amplification demonstrated using shortened cycling conditions.

For ultra-fast cycling, reverse transcription can be reduced to as little as 3 minutes at 47–52 °C, followed by polymerase activation at 95 °C for 1-2 minutes and 40 cycles of 95 °C for 1 second and 60 °C (combined annealing/extension) for 3 seconds. An optional pre-cycling step of 4 cycles of 95 °C for 1 second and 60 °C for 5 seconds may help improve amplification of challenging targets, but should be evaluated for each assay.

Ultra-fast cycling requires instruments capable of rapid temperature changes and efficient fluorescence acquisition. The Bio Molecular Systems Mic is particularly well suited to ultra-fast cycling, while instruments such as the Roche LightCycler® 480 and fast-mode Applied Biosystems™ QuantStudio™ systems also support accelerated cycling protocols.

The optimal conditions depend on the target, amplicon length, reaction complexity and instrument. We recommend using short amplicons of no more than 120 bp and verifying amplification efficiency and sensitivity when shortening the reverse transcription or cycling steps, particularly for multiplex assays and low-copy RNA targets. For UNG-containing mixes, include the additional UNG incubation specified in the product manual.