FAQs

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.

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.

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.

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.

The Clara® Probe Purple and Clara® Probe 1-Step Purple Mixes that contain passive reference dyes come in different formulations, each with a different concentration of the passive reference dye:

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 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.

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).

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).

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.