Clara® Probe 1-Step Mix

Ct values can vary depending on sample concentration, reaction optimization, equipment, and laboratories, so care should be taken when selecting a cutoff Ct value. Generally, Ct values above 35-40 are considered unreliable. However, late Cts can be observed for inefficient reactions using low sample copy numbers. It's always good practice to normalize cutoff values with relative or absolute quantification methods. Running and analyzing melt curves or gels of the product to identify products from any late amplification is also recommended.

Yes. Clara® Probe 1-Step Mix is capable of evenly amplifying cDNA and RNA targets. However, as this mix contains RTase, it is ideal for 1-step procedures. We recommend using Clara® Probe 1-Step Mix for 1-step procedures and when experiments require DNA detection in some limited samples and using Clara® Probe Mix for 2-step protocols and recurring DNA detection.

Yes, the PCR products generated with these mixes are characterized like those produced with wild-type Taq polymerase. They can be sequenced or digested with restriction enzymes using standard protocols. Products have 3′-d(A) overhangs and are suitable for TA cloning or can be cut or digested with restriction enzymes before cloning. For optimal results, we recommend purifying the PCR products with any standard PCR purification kit.

ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize for fluorescence level variations that may occur primarily due to optical changes between wells. The normalization of fluorescence intensity (Rn) is achieved in real-time PCR software by dividing the fluorescent emission intensity of the specific signal by that of ROX.

ROX does not participate in the PCR reaction, and its fluorescence intensity does not correlate with the amount of DNA per well; thus, adding this fluorophore to the mix provides a consistent fluorescence signal throughout the amplification process.

Different real-time PCR instrument types require different optimal ROX concentration passive reference standards, primarily due to varying optical configurations of each system (i.e., different excitation source and optics used).

Adding too little or too much ROX will result in noisy signals that affect the reaction results. Therefore, it is crucial for users to:

1. Identify the correct ROX concentration to optimize real-time PCR results, and
2. Check ROX settings in the software used to set up the reaction

A useful selection tool for frequently used systems can be found here.

No. Besides ROX (if present in the kit), there are no other dyes in our mixes. Thus, you can use any fluorophore-labeled probes for your reaction.

No, this mix contains an RNase inhibitor to prevent any degradation and enhance sensitivity.

Different products can generate different fluorescence levels. However, this does not affect the accuracy of quantification, and Ct values will not differ between products.

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

The Clara® Probe Purple and Clara® Probe 1-Step Purple mixes contain passive reference dyes paired with various formulations, each with different passive reference dye concentrations:

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

Separate-ROX (PB20.68 and PB25.88) mixes include a separate tube of 50 µM ROX additive. This allows you to choose the ROX concentration you wish to use.
You can use our qPCR Selection Tool under the Resources dropdown menu to identify which of our mixes best suits your qPCR machine.

ROX is a passive reference dye meaning it does not participate in the PCR reaction. It is used to normalize for non-PCR related fluorescence fluctuations. You can use our qPCR Selection Tool under the Resources section to determine which qPCR mix best suits your qPCR machine.

If inhibition is observed, the amount of sample in the reaction can be reduced. This will increase the Ct value but decrease the likelihood that inhibitors interfere with Taq DNA polymerase activity. If this is ineffective, try adding 0.4-4 mg/ml BSA to the reaction^1,2. Ensure the cycle conditions in our product guide are followed.

1. Kreader, C. A. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol 62, 1102-1106 (1996).
2. Wilson, I. G. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63, 3741-3751 (1997).

There are reports that efficiency can decrease with subsequent dilutions for the standard curve. We advise avoiding this by diluting standards in 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 0.05% Tween-20. EDTA is a chelating agent and plays a role in preventing DNAse activity¹. Tween-20 is a detergent and prevents DNA from sticking to the walls of tubes². Most microcentrifuge tubes are made from polypropylene, and research has shown DNA sticks very well to polypropylene³.

Standards should not be frozen after dilution. Even in the presence of detergent, freezing appears to cause DNA to stick irreversibly to polypropylene. We suggest keeping your standards at 4°C and preparing a fresh batch after several weeks.

1. Barra, G. B. et al. EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples. Clin Biochem 48, 976-981, doi:10.1016/j.clinbiochem.2015.02.014 (2015).
2. Linnarsson, S. Recent advances in DNA sequencing methods – general principles of sample preparation. Exp Cell Res 316, 1339-1343, doi:10.1016/j.yexcr.2010.02.036 (2010).
3. Gaillard, C. & Strauss, F. Avoiding adsorption of DNA to polypropylene tubes and denaturation of short DNA fragments. Technical Tips Online 3, 3 (1998).

There are several options to consider when optimizing the reaction:

• Decrease 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 DNA and using a 10x template dilution series. In addition to running these on a gel to see if non-specific products persist, the reaction's performance can be calculated with your qPCR machine's software after performing template dilution. If the performance falls within the range of 90 – 110%, the amplicon is doubling every cycle.

Higher Ct values typically indicate delayed amplification. This is likely due to too much template in the reaction causing primers and probes to bind to different DNA molecules. Templates usually contain a lot of non-target DNA and this can distract the oligos. We recommend diluting the templates (10x-1000x) to address this.

Furthermore, the annealing/extension temperature can also be increased to make oligo binding more specific to the target sequence and minimize background signal.

We recommend using 0.4 µM for each primer. There is some flexibility around this recommended concentration, however, the primer concentration should not be increased, as this can significantly affect the enzyme's activity.

For more information on multiplexing, please refer to our qPCR Technical Guide.

This is likely occurring because the time for the first step (hot start) is too short. Ensure that the hot start phase occurs at 95°C for 2 minutes to fully activate the enzyme. The recommended thermal profile is:

• 95°C (120 seconds)
• 40 cycles: 95°C (5-15 seconds) – 60°C (20-30 seconds)
• Melt curve

If non-specific products are still obtained, we recommend increasing the annealing/extension temperature from 60°C to 65°C, depending on the primer set used.

Yes, the 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 the following methods:

• Using universal RT primers and adding a poly(A) or poly(U) tail (e.g., with poly(U) polymerase enzyme), followed by cDNA synthesis with universal primers1,2.
• Using specific RT primers and skipping the tailing step 1, 3-5.

If you are unfamiliar with these methods, please refer to the list of publications below, which are considered guide references.

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).