Clara® Probe Mix

Ct values can vary between sample concentrations, reaction optimization, equipment, and laboratories, so caution is needed when choosing a Ct cut-off value. Generally, Ct values above 35-40 begin to be considered unreliable. However, late Ct values may be observed for inefficient reactions with low sample amounts. A good practice is to always standardize cut-off values using relative or absolute quantification methods. Also, it is advisable to run and analyze a melt curve or gel of the products to determine any late amplification products.

Yes, PCR products generated with these mixes have the characteristics of PCR products generated using wild-type Taq polymerase. They can be sequenced or digested using restriction endonucleases according to standard protocols. The product has a 3′-d(A) tail and can be used for TA cloning or can be blunted or digested with restriction enzymes prior to cloning. For best results, we recommend purifying PCR products using any standard PCR clean-up kit.

ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize fluctuating fluorescence levels mainly caused by optical changes between wells. The normalization of the fluorescence intensity (Rn) is performed in real-time PCR software by dividing the emission intensity of the specific signal by the emission intensity of ROX.

ROX does not participate in the PCR reaction and its fluorescence level does not correlate with the amount of DNA in each well, thus adding this fluorophore to the mix provides a constant fluorescence signal during amplification.

Different types of real-time PCR instruments require different optimal ROX concentrations for passive reference standards, mainly due to the different optical configurations of each system (i.e., different types of excitation sources and optics used).

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

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

A useful selection tool for the most commonly used systems can be found here.

No. Besides ROX (if included in the kit), there is no other dye in our mixes. You can use any fluorophore-labeled probe for your reaction.

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

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

Clara® Probe Purple and Clara® Probe 1-Step Purple mixes contain passive reference dyes with different formulations, each with a different concentration of passive reference dye: The Lo-ROX mixes (PB20.65 and PB25.85) contain 200 nM ROX.
The Hi-ROX mixes (PB20.66 and PB25.86) contain 2 µM ROX.
The No-ROX mixes (PB20.67 and PB25.87) contain no ROX. The Separate-ROX mixes (PB20.68 and PB25.88) include a separate tube containing 50 µM ROX additive. This allows you to choose the concentration of ROX you want to use.
You can use our qPCR Selection Tool in the Resources dropdown menu to determine which of our mixes are best suited for your qPCR machine.

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

If inhibition is observed, the sample amount in the reaction can be reduced. This will raise the Ct value but reduce the likelihood of inhibitors interfering with the activity of the Taq DNA polymerase. If this is not effective, try adding 0.4-4 mg/ml BSA to the reaction^1,2. Ensure that the cycling conditions in our product manual 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).

It has been reported that efficiency may decrease with subsequent dilutions for the standard curve. We recommend 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 it plays a role in preventing DNAse activity¹. Tween-20 is a detergent and prevents DNA from sticking to the sides of the tube². Most microcentrifuge tubes are made from polypropylene and research has shown that DNA adheres very strongly to polypropylene³.

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

1. Barra, G. B. et al. Inhibition caused by EDTA protects circulating 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. Online Tech Tips 3, 3 (1998).

There are multiple options to consider when optimizing the reaction:

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

Dilute the DNA template by starting with 5 ng DNA and using a 10-fold serial dilution of the sample. In addition to running these on a gel to see if non-specific products exist, the efficiency of the reaction can be calculated using the qPCR instrument software following the serial dilution. If the efficiency is between 90 – 110%, the amplicon is doubling each cycle.

Higher Ct values typically indicate slow amplification. This could primarily be due to an excess of template in the reaction, resulting in the primers and probe binding to different DNA molecules. Samples typically contain a lot of DNA beyond the target gene and this can scatter the oligos. We advise diluting the samples (10x-1000x) to address this issue.

Furthermore, the annealing/extension temperature can also be raised to make the oligo binding more specific to the target sequence and reduce background signals.

We recommend using 0.4 µM for each primer. There is some flexibility around this recommended concentration, however, primer concentrations should not be increased, as this may significantly impact enzyme activity.

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

This is most likely due to too short a time for the 1^st step (hot start). Ensure that the hot start phase is performed at 95°C for 2 minutes to fully activate the enzyme. The recommended temperature profile is:

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

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