Clara™ Probe 1-Step Mix

RT-qPCRs are no longer a challenge. Get clear and consistent results every time with Clara™ Probe 1-Step Mix. This new single tube RT-qPCR mix is suitable for universal probe-based detection of RNA and DNA targets in single and multiplex assays.

This carefully balanced 4x RT-qPCR mix combines maximum sensitivity with ease-of-use for streamlined 1-step probe RT-qPCR workflows. As a result, the mix shows no loss of efficiency in amplifying DNA or RNA and is ideal for users who detect both types of targets without the requirement for separate qPCR reagents.

Features

  • Concentrated 4x mix format, ideal for high-throughput, highly multiplexed assays
  • Ultra-sensitive detection of RNA & DNA targets
  • Early detection of a wide range of template amounts
  • Contains modified UltraScript™ RTase in a single-tube format
  • Advanced RNase inhibitor
  • Antibody-mediated hot start technology
  • Compatible with all real-time PCR platforms – standard and fast cycling conditions
CAT No.
Product
Size
Price
QTY
PB25.81-01
Clara™ Probe 1-Step Mix Lo-ROX
200 x 20 μL Reactions
$305.00
PB25.81-03
Clara™ Probe 1-Step Mix Lo-ROX
600 x 20 μL Reactions
$837.00
PB25.81-05
Clara™ Probe 1-Step Mix Lo-ROX
1000 x 20 μL Reactions (5 x 1 mL)
$1,200.00
PB25.81-50
Clara™ Probe 1-Step Mix Lo-ROX
10 000 x 20 μL Reactions (1 x 50 mL)
PB25.82-01
Clara™ Probe 1-Step Mix Hi-ROX
200 x 20 μL Reactions
$305.00
PB25.82-03
Clara™ Probe 1-Step Mix Hi-ROX
600 x 20 μL Reactions
$837.00
PB25.82-05
Clara™ Probe 1-Step Mix Hi-ROX
1000 x 20 μL Reactions (5 x 1 mL)
$1,200.00
PB25.82-50
Clara™ Probe 1-Step Mix Hi-ROX
10 000 x 20 μL Reactions (1 x 50 mL)
PB25.83-01
Clara™ Probe 1-Step Mix No-ROX
200 x 20 μL Reactions
$305.00
PB25.83-03
Clara™ Probe 1-Step Mix No-ROX
600 x 20 μL Reactions
$837.00
PB25.83-05
Clara™ Probe 1-Step Mix No-ROX
1000 x 20 μL Reactions (5 x 1 mL)
$1,200.00
PB25.83-50
Clara™ Probe 1-Step Mix No-ROX
10 000 x 20 μL Reactions (1 x 50 mL)
PB25.84-01
Clara™ Probe 1-Step Mix Separate-ROX
200 x 20 μL Reactions
$305.00
PB25.84-03
Clara™ Probe 1-Step Mix Separate-ROX
600 x 20 μL Reactions
$837.00
PB25.84-05
Clara™ Probe 1-Step Mix Separate-ROX
1000 x 20 μL Reactions (5 x 1 mL)
$1,200.00

To view your price please login or contact us

More Information

Probe 1-step qPCR mix – Clear Results, Reliable Conclusions

Clara™ Probe 1-Step Mix enables qPCR and 1-step RT-qPCR with the highest sensitivity, reliability, and with the greatest ease of use in diagnostic applications and basic research alike. It is a universal qPCR mix suitable for all types of probe technologies, including TaqMan®, Scorpions® and molecular beacons. This is our first generation of all-in-one 1-step RT-qPCR mixes, where both the DNA polymerase and reverse transcriptase are included in the same mastermix. Clara™ Probe 1-Step Mix is powered by our unique hot start Taq DNA polymerase and a specially modified version of our high performance, thermostable UltraScript™ Reverse Transcriptase. Rigorous testing during development makes this mix highly suitable for both single and multiplex detection with high efficiency and sensitive detection whether you’re detecting one target or many simultaneously.

Get the greatest ease of use

Our proprietary buffer composition (identified via smart screen technology) and uniquely modified UltraScript™ RTase allow the mix to be stored in a single tube, as all-in-one, RT-qPCR mastermix. This reduces the amount of pipetting needed for assay setup, minimising the risk of contamination, and saving time. It also means that one reagent is suitable for amplification of DNA and RNA and both target types can be included on the same plate or in the same run.  Having one mix means you do not require a different setup when primarily investigating RNA targets, but still requiring DNA quantification.

While users who test large quantities of both target types of targets may prefer dedicated reagents for each target type, Clara™ Probe Mix is developed exclusively for DNA and cDNA probe-based detection.

Improve reliability of real time PCR

Extensive optimization makes this mix suitable for all nucleic acid target types. We have tested it against common RNA viruses, including SARS-CoV-2, RSV, Influenza A, and B, standard housekeeping genes, such as g-actin and GAPDH, as well as DNA targets. The presence of RTase in the mix does not affect direct amplification of DNA targets, meaning you get the same high efficiency whether you’re detecting DNA or RNA sequences. The mix is highly stable and shows strong reproducibility between runs, giving your data reproducibility and confidence in the experimental conclusions you draw.

Maximise 1-step qPCR sensitivity

The high concentration 4x mix format of Clara™ Probe 1-Step Mix offers greater sensitivity and flexibility by allowing more sample to be added to each reaction, and for smaller reaction volumes to be used with confidence. Fast amplification allows for earlier Cts and permits reliable detection of down to four target copies per 20 μL reaction (0.8 copies per μL). Get the superior detection even in the most dilute samples.

Applications

  • Gene expression analysis
  • Genotyping
  • Allelic discriminiation
  • In vitro diagnostic kit development
  • Single & multiplex RNA & DNA detection

Specifications

Clara™ Probe 1-Step Mix Lo-ROX

Component

200 Reactions

600 Reactions

1000 Reactions

10 000 Reactions

4x Clara™ Probe 1-Step Mix Lo-ROX

1 x 1 mL

3 x 1 mL

5 x 1 mL

1 x 50 mL

Clara™ Probe 1-Step Mix Hi-ROX

Component

200 Reactions

600 Reactions

1000 Reactions

10 000 Reactions

4x Clara™ Probe 1-Step Mix Hi-ROX

1 x 1 mL

3 x 1 mL

5 x 1 mL

1 x 50 mL

Clara™ Probe 1-Step Mix Νo-ROX

Component

200 Reactions

600 Reactions

1000 Reactions

10 000 Reactions

4x Clara™ Probe 1-Step Mix Νo-ROX

1 x 1 mL

3 x 1 mL

5 x 1 mL

1 x 50 mL

Clara™ Probe 1-Step Mix Separate-ROX

Component

200 Reactions

600 Reactions

1000 Reactions

4x Clara™ Probe 1-Step Mix Νo-ROX

1 x 1 mL

3 x 1 mL

5 x 1 mL

50 μΜ ROX Additive

1 x 200 μL

1 x 200 μL

1 x 200 μL

Clara™ Probe 1-Step Mix Lo-ROX

Component

4x Clara™ Probe 1-Step Mix Lo-ROX



200 Reactions

1 x 1 mL

600 Reactions

3 x 1 mL

1000 Reactions

5 x 1 mL

10 000 Reactions

1 x 50 mL

Clara™ Probe 1-Step Mix Hi-ROX

Component

4x Clara™ Probe 1-Step Mix Hi-ROX



200 Reactions

1 x 1 mL

600 Reactions

3 x 1 mL

1000 Reactions

5 x 1 mL

10 000 Reactions

1 x 50 mL

Clara™ Probe 1-Step Mix Νo-ROX

Component

4x Clara™ Probe 1-Step Mix Νo-ROX



200 Reactions

1 x 1 mL

600 Reactions

3 x 1 mL

1000 Reactions

5 x 1 mL

10 000 Reactions

1 x 50 mL

Clara™ Probe 1-Step Mix Separate-ROX

Component

4x Clara™ Probe 1-Step Mix Νo-ROX

50 μΜ ROX Additive



200 Reactions

1 x 1 mL

1 x 200 μL

600 Reactions

3 x 1 mL

1 x 200 μL

1000 Reactions

5 x 1 mL

1 x 200 μL

Reaction Volume

Storage

20 μL

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

20 μL

Storage

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

FAQs

Beyond what Ct value are results unreliable?

Ct values can vary between template concentrations, reaction optimisation, instruments and laboratories, so care must be taken when selecting a cut-off Ct value. Generally, Ct values over 35-40 would begin to be considered unreliable. However, late Cts could be observed for inefficient reactions using low template copy number. It is always good practice to normalise cut-offs with relative or absolute quantification methods. It is also recommended to run and analyse melt curves or gels of the products to determine products of any late amplifications.

Can Clara™ Probe 1-Step Mix be used for both 1-step and 2-step RT-PCR?

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.

Can products generated with Clara™ Probe Mix and Clara™ Probe 1-Step Mix be digested, cloned, and sequenced?

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.

Can ROX have a negative impact on the reaction?

ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize for variations of fluorescence levels that can arise mainly due to optical path variations among wells. Normalisation of the fluorescence intensity (Rn) is done in real-time PCR software by dividing the emission intensity of the specific signal by the emission intensity of ROX.

ROX does not take part in the PCR reaction and its fluorescence levels are not proportional to the quantity of DNA in each well, so the addition of this fluorophore to a mix provides a constant fluorescent signal during amplification.

Different types of real-time PCR instruments requiring a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system (i.e. the different type of excitation source and optics used).

The addition of either too little or too much ROX would result in a very noisy signal impacting on the results of the reaction. Therefore, it is extremely important for the user to:

  1. Determine the correct ROX concentration to optimise 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.

Do Clara™ Probe Mix and Clara™ Probe 1-Step Mix contain the FAM dye in it?

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.

Do I need to use an RNase inhibitor in my reactions?

No, the mix contains an RNase inhibitor to prevent any degradation and increase sensitivity.

Is it normal if the fluorescence of Clara™ Probe Mix and Clara™ Probe 1-Step Mix differs from the one obtained with competitors’ products?

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.

Is the storage of sample DNA in 1x TE (10 mM Tris-HCl / 1 mM EDTA) buffer compatible with subsequent qPCR using Clara™ Probe Mix and Clara™ Probe 1-Step Mix?

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.

What are the ROX concentrations in Clara™ Probe Mix and Clara™ Probe 1-Step Mix?

The qPCRBIO Probe Mixes that contain passive reference dyes come in different formulations, each with a different concentration of the passive reference dye:

 

Lo-ROX mixes (PB20.51 and PB25.81) contain 200 nM ROX.

Hi-ROX mixes (PB20.52 and PB25.82) contain 2 µM ROX.

No-ROX mixes (PB20.53 and PB25.83) do not contain ROX.

Separate-ROX mixes (PB20.54 and PB25.84) 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.

What is ROX and do I need it?

ROX is a passive reference dye which means it does not take part in the PCR reaction. It is used to normalise non-PCR related fluctuations in fluorescence. 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.

What should I do if the reaction is inhibited?

If inhibition is observed, the amount of template in the reaction can be decreased. This will increase the Ct value but lower the likelihood of inhibitors interfering with the Taq DNA polymerase activity. If this doesn’t work, try adding 0.4-4 mg/ml of BSA to the reaction1,2. Ensure the cycling conditions in our product manual are adhered to.

 

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).

What should I do when amplification efficiency reduces with serial dilutions?

It has been reported that efficiency can decrease with subsequent dilutions for the standard curve. We recommend avoiding this by diluting the 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 the DNA from adsorbing to the sides of the tubes2. Most microcentrifuges are made of polypropylene and research has demonstrated that DNA sticks very well to polypropylene3.

 

Standards should not be frozen after diluting them. Even in the presence of detergent, freezing seems to cause DNA to bind irreversibly to polypropylene. We suggest leaving your standards at 4°C and preparing a fresh batch every few 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).

What troubleshooting is recommended if there are non-specific products using Clara™ Probe Mix and Clara™ Probe 1-Step Mix?

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.

What troubleshooting is recommended when Ct values are higher than usual?

Higher Ct values are generally indicative of delayed amplification. This could most likely be due to an excess of template in the reaction resulting in the primers and probes being bound on different DNA molecules. Samples usually have a lot of DNA other than the target gene and this can scatter the oligos. We recommend diluting the samples (10x-1000x) to solve this.

Moreover, annealing/extension temperature can also be increased to make the binding of the oligos more specific to the target sequence and decrease background signal.

When performing a multiplex, what is the recommended concentration of each primer?

We recommend using 0.4 µM of each primer. There is a degree of flexibility around this recommended concentration however, the primer concentration should not be increased, as this may significantly affect the activity of the enzyme.

For further considerations on multiplexing please refer to our qPCR Technical Guide.

Why is there a non-specific product when using the same primers and PCR conditions as a competitor’s product?

It’s most likely because the time for the 1st step (hot start) is too short. Ensure that the hot start phase is done 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

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

Will this mix work for micro RNA templates?

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).

More Information

Probe 1-step qPCR mix – Clear Results, Reliable Conclusions

Clara™ Probe 1-Step Mix enables qPCR and 1-step RT-qPCR with the highest sensitivity, reliability, and with the greatest ease of use in diagnostic applications and basic research alike. It is a universal qPCR mix suitable for all types of probe technologies, including TaqMan®, Scorpions® and molecular beacons. This is our first generation of all-in-one 1-step RT-qPCR mixes, where both the DNA polymerase and reverse transcriptase are included in the same mastermix. Clara™ Probe 1-Step Mix is powered by our unique hot start Taq DNA polymerase and a specially modified version of our high performance, thermostable UltraScript™ Reverse Transcriptase. Rigorous testing during development makes this mix highly suitable for both single and multiplex detection with high efficiency and sensitive detection whether you’re detecting one target or many simultaneously.

Get the greatest ease of use

Our proprietary buffer composition (identified via smart screen technology) and uniquely modified UltraScript™ RTase allow the mix to be stored in a single tube, as all-in-one, RT-qPCR mastermix. This reduces the amount of pipetting needed for assay setup, minimising the risk of contamination, and saving time. It also means that one reagent is suitable for amplification of DNA and RNA and both target types can be included on the same plate or in the same run.  Having one mix means you do not require a different setup when primarily investigating RNA targets, but still requiring DNA quantification.

While users who test large quantities of both target types of targets may prefer dedicated reagents for each target type, Clara™ Probe Mix is developed exclusively for DNA and cDNA probe-based detection.

Improve reliability of real time PCR

Extensive optimization makes this mix suitable for all nucleic acid target types. We have tested it against common RNA viruses, including SARS-CoV-2, RSV, Influenza A, and B, standard housekeeping genes, such as g-actin and GAPDH, as well as DNA targets. The presence of RTase in the mix does not affect direct amplification of DNA targets, meaning you get the same high efficiency whether you’re detecting DNA or RNA sequences. The mix is highly stable and shows strong reproducibility between runs, giving your data reproducibility and confidence in the experimental conclusions you draw.

Maximise 1-step qPCR sensitivity

The high concentration 4x mix format of Clara™ Probe 1-Step Mix offers greater sensitivity and flexibility by allowing more sample to be added to each reaction, and for smaller reaction volumes to be used with confidence. Fast amplification allows for earlier Cts and permits reliable detection of down to four target copies per 20 μL reaction (0.8 copies per μL). Get the superior detection even in the most dilute samples.

Applications

  • Gene expression analysis
  • Genotyping
  • Allelic discriminiation
  • In vitro diagnostic kit development
  • Single & multiplex RNA & DNA detection

Specifications

Clara™ Probe 1-Step Mix Lo-ROX

Component

200 Reactions

600 Reactions

1000 Reactions

10 000 Reactions

4x Clara™ Probe 1-Step Mix Lo-ROX

1 x 1 mL

3 x 1 mL

5 x 1 mL

1 x 50 mL

Clara™ Probe 1-Step Mix Hi-ROX

Component

200 Reactions

600 Reactions

1000 Reactions

10 000 Reactions

4x Clara™ Probe 1-Step Mix Hi-ROX

1 x 1 mL

3 x 1 mL

5 x 1 mL

1 x 50 mL

Clara™ Probe 1-Step Mix Νo-ROX

Component

200 Reactions

600 Reactions

1000 Reactions

10 000 Reactions

4x Clara™ Probe 1-Step Mix Νo-ROX

1 x 1 mL

3 x 1 mL

5 x 1 mL

1 x 50 mL

Clara™ Probe 1-Step Mix Separate-ROX

Component

200 Reactions

600 Reactions

1000 Reactions

4x Clara™ Probe 1-Step Mix Νo-ROX

1 x 1 mL

3 x 1 mL

5 x 1 mL

50 μΜ ROX Additive

1 x 200 μL

1 x 200 μL

1 x 200 μL

Clara™ Probe 1-Step Mix Lo-ROX

Component

4x Clara™ Probe 1-Step Mix Lo-ROX



200 Reactions

1 x 1 mL

600 Reactions

3 x 1 mL

1000 Reactions

5 x 1 mL

10 000 Reactions

1 x 50 mL

Clara™ Probe 1-Step Mix Hi-ROX

Component

4x Clara™ Probe 1-Step Mix Hi-ROX



200 Reactions

1 x 1 mL

600 Reactions

3 x 1 mL

1000 Reactions

5 x 1 mL

10 000 Reactions

1 x 50 mL

Clara™ Probe 1-Step Mix Νo-ROX

Component

4x Clara™ Probe 1-Step Mix Νo-ROX



200 Reactions

1 x 1 mL

600 Reactions

3 x 1 mL

1000 Reactions

5 x 1 mL

10 000 Reactions

1 x 50 mL

Clara™ Probe 1-Step Mix Separate-ROX

Component

4x Clara™ Probe 1-Step Mix Νo-ROX

50 μΜ ROX Additive



200 Reactions

1 x 1 mL

1 x 200 μL

600 Reactions

3 x 1 mL

1 x 200 μL

1000 Reactions

5 x 1 mL

1 x 200 μL

Reaction Volume

Storage

20 μL

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

Reaction Volume

20 μL

Storage

On arrival, products should be stored between -30 and -15°C. If stored correctly the kit will retain full activity for 12 months.

FAQs

Beyond what Ct value are results unreliable?

Ct values can vary between template concentrations, reaction optimisation, instruments and laboratories, so care must be taken when selecting a cut-off Ct value. Generally, Ct values over 35-40 would begin to be considered unreliable. However, late Cts could be observed for inefficient reactions using low template copy number. It is always good practice to normalise cut-offs with relative or absolute quantification methods. It is also recommended to run and analyse melt curves or gels of the products to determine products of any late amplifications.

Can Clara™ Probe 1-Step Mix be used for both 1-step and 2-step RT-PCR?

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.

Can products generated with Clara™ Probe Mix and Clara™ Probe 1-Step Mix be digested, cloned, and sequenced?

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.

Can ROX have a negative impact on the reaction?

ROX (6-carboxy-X-rhodamine) is used as a passive reference dye in ROX-dependent real-time PCR instruments to normalize for variations of fluorescence levels that can arise mainly due to optical path variations among wells. Normalisation of the fluorescence intensity (Rn) is done in real-time PCR software by dividing the emission intensity of the specific signal by the emission intensity of ROX.

ROX does not take part in the PCR reaction and its fluorescence levels are not proportional to the quantity of DNA in each well, so the addition of this fluorophore to a mix provides a constant fluorescent signal during amplification.

Different types of real-time PCR instruments requiring a passive reference standard have different optimal concentrations of ROX, mainly due to the different optical configurations of each system (i.e. the different type of excitation source and optics used).

The addition of either too little or too much ROX would result in a very noisy signal impacting on the results of the reaction. Therefore, it is extremely important for the user to:

  1. Determine the correct ROX concentration to optimise 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.

Do Clara™ Probe Mix and Clara™ Probe 1-Step Mix contain the FAM dye in it?

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.

Do I need to use an RNase inhibitor in my reactions?

No, the mix contains an RNase inhibitor to prevent any degradation and increase sensitivity.

Is it normal if the fluorescence of Clara™ Probe Mix and Clara™ Probe 1-Step Mix differs from the one obtained with competitors’ products?

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.

Is the storage of sample DNA in 1x TE (10 mM Tris-HCl / 1 mM EDTA) buffer compatible with subsequent qPCR using Clara™ Probe Mix and Clara™ Probe 1-Step Mix?

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.

What are the ROX concentrations in Clara™ Probe Mix and Clara™ Probe 1-Step Mix?

The qPCRBIO Probe Mixes that contain passive reference dyes come in different formulations, each with a different concentration of the passive reference dye:

 

Lo-ROX mixes (PB20.51 and PB25.81) contain 200 nM ROX.

Hi-ROX mixes (PB20.52 and PB25.82) contain 2 µM ROX.

No-ROX mixes (PB20.53 and PB25.83) do not contain ROX.

Separate-ROX mixes (PB20.54 and PB25.84) 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.

What is ROX and do I need it?

ROX is a passive reference dye which means it does not take part in the PCR reaction. It is used to normalise non-PCR related fluctuations in fluorescence. 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.

What should I do if the reaction is inhibited?

If inhibition is observed, the amount of template in the reaction can be decreased. This will increase the Ct value but lower the likelihood of inhibitors interfering with the Taq DNA polymerase activity. If this doesn’t work, try adding 0.4-4 mg/ml of BSA to the reaction1,2. Ensure the cycling conditions in our product manual are adhered to.

 

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).

What should I do when amplification efficiency reduces with serial dilutions?

It has been reported that efficiency can decrease with subsequent dilutions for the standard curve. We recommend avoiding this by diluting the 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 the DNA from adsorbing to the sides of the tubes2. Most microcentrifuges are made of polypropylene and research has demonstrated that DNA sticks very well to polypropylene3.

 

Standards should not be frozen after diluting them. Even in the presence of detergent, freezing seems to cause DNA to bind irreversibly to polypropylene. We suggest leaving your standards at 4°C and preparing a fresh batch every few 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).

What troubleshooting is recommended if there are non-specific products using Clara™ Probe Mix and Clara™ Probe 1-Step Mix?

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.

What troubleshooting is recommended when Ct values are higher than usual?

Higher Ct values are generally indicative of delayed amplification. This could most likely be due to an excess of template in the reaction resulting in the primers and probes being bound on different DNA molecules. Samples usually have a lot of DNA other than the target gene and this can scatter the oligos. We recommend diluting the samples (10x-1000x) to solve this.

Moreover, annealing/extension temperature can also be increased to make the binding of the oligos more specific to the target sequence and decrease background signal.

When performing a multiplex, what is the recommended concentration of each primer?

We recommend using 0.4 µM of each primer. There is a degree of flexibility around this recommended concentration however, the primer concentration should not be increased, as this may significantly affect the activity of the enzyme.

For further considerations on multiplexing please refer to our qPCR Technical Guide.

Why is there a non-specific product when using the same primers and PCR conditions as a competitor’s product?

It’s most likely because the time for the 1st step (hot start) is too short. Ensure that the hot start phase is done 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

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

Will this mix work for micro RNA templates?

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).