Yes, PCR products generated with qPCRBIO SyGreen 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.

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.

Yes. We have customers who have reported successful use of qPCRBIO SyGreen Mix on bisulphate converted gDNA.

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.

SyGreen belongs to the family of asymmetric cyanine dyes, which is the same family that SYBR™ Green belongs to. SyGreen is an improved, new generation dye from this family. Similar to SYBR™ Green, SyGreen intercalates the DNA. Unlike SYBR™ Green, SyGreen supports PCR amplification over a wide range of dye concentrations, that is, the dye does not inhibit the PCR reaction. This plays a role in producing robust and consistent DNA melting curves both with prokaryote and eukaryote templates, as it shows no preference in nucleotide composition.

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

• qPCRBIO SyGreen Mix Lo-ROX (PB20.11) contains 112 nM ROX.
• qPCRBIO SyGreen Mix Hi-ROX (PB20.12) contains 1.12 µM ROX.
• qPCRBIO SyGreen Mix Fluorescein (PB20.13) contains 0.2 mM fluorescein.
• qPCRBIO SyGreen Mix Separate-ROX (PB20.14) 2x mix contains no ROX and 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.

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

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.

The enzyme has an error rate of approximately 1 error per 2.0 x 10⁵ nucleotides incorporated.

All qPCRBIO SyGreen Mixes contain MgCl₂ at a concentration of 6 mM. This means the final concentration in the reaction is 3 mM.

Ensure you are using the right concentration of ROX because different instruments require different ROX concentrations. For example, if a qPCRBIO SyGreen 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.

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).

A high background level of fluorescence is most likely due to an excess of template in the reaction. This relates to the qPCR instrument picking up the SyGreen dye which binds to the template DNA. Samples usually contain a lot of DNA other than the target gene, so there will be enough present to register fluorescence. We recommend diluting the samples 100x-1000x to overcome this issue.

This should not only reduce the background signal, but also allow an accurate quantification because the dilution should bring the Ct values in the range of those obtained with the standards. Keep in mind that if the Ct is earlier than the first standard, it won’t be considered accurate enough quantification for publication.

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.

An early Ct could indicate one of two things. The first could be the result of our product performing very well relative to the competitor’s product. We have designed our products to be faster than our competitors, and we have found that we are significantly faster than many.

The second could be that caused by non-specific products. This can be verified by running a melt analysis at the end of the qPCR run. If your results show one peak, it is likely the correct product was amplified. You may observe the position of the peak being different with our mix relative to our competitor’s mix but is this normal because the salt concentration and pH affect the melting temperature. If your results show more than one peak, then the reaction is not specific. PCR products can also be run on an agarose gel to verify if there is only one band, and if it is the same size as the amplicon obtained with the competitor’s product.

To address the issue of a non-specific product, the thermal cycling will need to be adjusted.