<aside>

Designing a hydrolysis probe assay for Countable PCR? Choosing the right fluorophore and quencher improves signal, reduces background, and improves counting accuracy. . This quick guide helps you pick the optimal combinations of fluorophores and quenchers for your design—such that you get good assay signal and noise at the get-go. .

</aside>

Recommended fluorophores

Each Countable channel is optimized for a specific set of fluorophores based on spectral characteristics. You can use any fluorophore in the compatible list for each channel, but the recommended option will typically give you the best signal-to-noise ratio.

Recommended quenchers

Hydrolysis probes for Countable PCR are always designed with a 5’ fluorophore and a 3’ quencher. Additionally, we always recommend the inclusion of a probe additive to suppress background fluorescence. Ensure you use the compatible quencher paired with each fluorophore selected for the channel, for both the probe additive (PA). Internal quenchers (e.g. ZEN or TAO, from IDT) are compatible, although not needed when PA is included in the assay.

Tips

• Use a probe additive (PA) - PAs reduce background and improve counting accuracy. We strongly recommend including them in any hydrolysis probe assay.
• Purify with HPLC - All probes and PAs should be HPLC-purified to ensure quality.
• Skip MGB probes - ****Use LNA instead—they give lower background on the Countable Platform.

Next steps

• Use the PA Designer to design your probe additive

<aside>

When designing your Countable assay, picking the right fluorophore–quencher pair matters. It ensures optimal signal to noise performance —and gets you closer to results you can trust. Once you've chosen your probe chemistry, your next step is designing a probe additive.

</aside>