Clara® Probe 1-Step Purple Mix

Yes. Clara® Probe 1-Step Mix can amplify target cDNA and RNA equally efficiently. However, as the mix contains RTase, it is ideal for a one-step procedure. We recommend using Clara® Probe 1-Step Mix for one-step procedures and when experiments call for DNA detection in a limited number of samples, and Clara® Probe Mix for two-step protocols and routine DNA detection.

Yes, the PCR products generated from these mixes have attributes identical to PCR products generated from wild-type Taq polymerase. They can be decoded or resolved with standard restriction endonucleases. The products have 3′-d(A) overhangs and can be used for TA cloning or trimmed blunt or resolved with restriction enzymes before cloning. For best results, we recommend purifying the PCR products using any standard PCR purification kit.

No. Besides ROX (if in kit), there are no other dyes in our mixes. Therefore, you can use any fluorophore-linked probe for your reaction.

Different products may yield different fluorescence levels. However, this does not affect the accuracy of quantification, and the Ct values will not be significantly different between products.

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

Clara® Probe Purple Mixes and Clara® Probe 1-Step Purple Mixes contain passive reference dye in several formulations, each with different passive reference dye concentrations:

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 allows you to choose the ROX concentration you want to use.
You can use our qPCR Selection Tool under the Resources menu to determine which of our mixes is best aligned with your qPCR machine.

There are several options to consider when optimizing the reaction:

• Reduce the annealing/extension time to 5 seconds
• Raise the annealing/extension temperature from 60 to 65°C

Dilute initial DNA sample with 5ng DNA and use a 10x sample dilution series. Besides running these on a gel to see if non-specific products persist, reaction performance can be calculated with the qPCR machine’s software after performing the sample dilution series. If the efficiency is between 90 – 110%, the amplicon is doubling each cycle.

Yes, Clara® Probe 1-Step Mix can be used for micro RNA samples. Although we do not sell dedicated kits, all our RTases can be used for miRNA quantification and analysis.

We recommend using one of two methods:

• Use general RT primers and append poly(A) or poly(U) tails (e.g., using poly(U)-polymerase), then perform cDNA synthesis using general primers1,2.
• Use specific RT primers and skip the tailing step1, 3-5.

If unfamiliar with the details of those methods, please refer to the literature list below, serving as a guide.

1. Dave, V. P. et al. Amplification and detection technologies for microRNA: 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. An easy and specific assay for microRNA quantification using an optimized RT-qPCR method. 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).