FAQs

Yes. Clara™ Probe 1-Step Mix can amplify cDNA and RNA targets equally well. However, since this mix contains RTase, it is ideal for 1-step workflows. We recommend using Clara™ Probe 1-Step Mix for 1-step workflows and where experiments require DNA detection in a limited number of samples, and Clara™ Probe Mix for 2-step protocols and routine DNA detection.

Yes, PCR products generated with these mixes have the same characteristics as PCR products generated with wild-type Taq polymerase. They may be sequenced or digested with restriction endonucleases using standard protocols. Products are 3′-d(A)-tailed and may be used for TA cloning or may be blunt-ended or digested with restriction enzymes prior to cloning. For best results, we recommend purifying the PCR products using any standard PCR clean-up kit.

No. Apart from ROX (if present in the kit), there is no other dye in our mixes. You can therefore use any fluorophore-conjugated probe for your reaction.

Different products could give a different plateau of fluorescence. However, this has no impact on quantification accuracy and Ct values will not differ among products.

Yes, this storage buffer is compatible. The EDTA will chelate some of the magnesium in the mix, but not significantly enough to affect the reaction.

The Clara™ Probe Purple and Clara™ Probe 1-Step Purple Mixes that contain passive reference dyes come in different formulations, each with a different concentration of the passive reference dye:

Lo-ROX mixes (PB20.65 and PB25.85) contain 200 nM ROX.
Hi-ROX mixes (PB20.66 and PB25.86) contain 2 µM ROX.
No-ROX mixes (PB20.67 and PB25.87) do not contain ROX.

Separate-ROX mixes (PB20.68 and PB25.88) include a separate tube of 50 µM ROX additive. This enables you to choose what concentration of ROX you’d like to use.
You can use our qPCR Selection Tool under the Resources drop-down menu to determine which of our mixes are best suited for your qPCR machine.

There are different options to consider when optimising the reaction:

Reduce the annealing/extension time to 5 seconds

Increase the annealing/extension temperature from 60 to 65°C

Dilute the DNA template by starting with 5ng of DNA and using a 10x template dilution series. In addition to running these on a gel to see if the non-specific products persist, the efficiency of the reaction can be calculated with the software of the qPCR instrument after doing the template dilution. If the efficiency is between 90 – 110%, then the amplicon is being doubled every cycle.

Yes, Clara Probe 1-Step Mix can be used for micro RNA templates. Although we do no sell dedicated kits, all of our RTases can be used for miRNA quantification and analysis.

We advise that you use one of the two following approaches:

Use universal RT primers and add poly(A) or poly(U) tails (e.g. by poly(U)-polymerase), followed by cDNA synthesis using universal primers1,2.

Use specific RT primers and omit the tailing step1,3-5.

If you are unfamiliar with the specifics of those approaches, please refer to the reference list below, which serve as a guideline.

1  Dave, V. P. et al. MicroRNA amplification and detection technologies: opportunities and challenges for point of care diagnostics. Lab Invest 99, 452-469, doi:10.1038/s41374-018-0143-3 (2019).

2  Mei, Q. et al. A facile and specific assay for quantifying microRNA by an optimized RT-qPCR approach. PLoS One 7, e46890, doi:10.1371/journal.pone.0046890 (2012).

3  Chen, C. et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33, e179, doi:10.1093/nar/gni178 (2005).

4  Raymond, C. K., Roberts, B. S., Garrett-Engele, P., Lim, L. P. & Johnson, J. M. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11, 1737-1744, doi:10.1261/rna.2148705 (2005).

5  Androvic, P., Valihrach, L., Elling, J., Sjoback, R. & Kubista, M. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Res 45, e144, doi:10.1093/nar/gkx588 (2017).