FAQs qPCRBIO Probe Mix – Witec AG

qPCRBIO Probe Mixes are ready to use qPCR 2x Mastermixes. You only need to add primers, template DNA and PCR grade water during reaction set up.

What is ROX?

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.

What is the concentration of dNTPs in qPCRBIO Probe Mixes?

All qPCRBIO Probe Mixes contain dNTPs at a concentration of 2 mM (0.5 mM each). This means the final concentration in the reaction is 1 mM (0.25 mM each).

What is the MgCl2 concentration in qPCRBIO Probe Mixes?

All qPCRBIO Probe Mixes contain MgCl₂ at a concentration of 12 mM. This means the final concentration in the reaction is 6 mM.

What should be considered if the normalisation of signal with ROX appears to be lower relative to a competitor’s mix?

Ensure you are using the right concentration of ROX because different instruments require different ROX concentrations. For example, if a qPCRBIO Probe Mix Hi-ROX is used with an instrument requiring a Lo-ROX mix, the software will normalise the qPCRBIO signal against the Hi-ROX level. This will significantly reduce the fluorescence level of the qPCRBIO mix relative to competitors’ mixes, if in that case the Lo-ROX mix was chosen for the competitor.

When comparing mixes from different manufacturers, it is better to carry out separate runs or turn the passive reference off before analysing data.

What troubleshooting is recommended if efficiency of amplification is reduced with standard 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 the reaction is being 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 reaction^1,2. 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 troubleshooting is recommended if there are non-specific products using qPCRBIO Probe Mixes?

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.

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.

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 1^st 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.