FAQs

We have not screened for any other components to add to the Lyo-Ready Inhibitor-Tolerant Probe (1-Step) Mix apart from primers, probes and ROX. If you would like to add extra lyo-compatible components, you will need to test their activity in both the wet mix and the lyophilised material, as well as assess their stability over the time. PCR Biosystems is not responsible for any loss of performance caused by the added component.

Yes, ROX (6-carboxy-X-rhodamine), available separately, can be added to the kit and it would not interfere with the lyophilisation process.

ROX 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 template 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.

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, results are still accurate even with reduced fluorescence. We have observed even 10-fold reduction in the fluorescence plateau when amplifying the same target in crude blood versus extracted samples. However, there is no inhibition of the mix up to the levels we have tested (6.25% crude blood:EDTA), this can be verified either by normalising fluorescence between crude blood and other samples, or by modifying the threshold used so that it is always the same percentage of the max fluorescence reached (e.g., 10%).

4x Lyo-Ready Probe Mix contains MgCl₂ at a concentration of 18 mM. This means the final concentration in the reaction is 4.5 mM.

In the product manual and associated drying guide we provide a general lyophilisation protocol, tested for 2 mL glass vials filled with 500 μL of the Lyo-Ready (Inhibitor-Tolerant) Probe (1-Step) Mix. We observed that lyophilisation is facilitated (i.e., the program is shorter, and cakes have a more uniform aspect with no signs of shrinkage) when the Lyo-Ready (Inhibitor-Tolerant) Probe (1-Step) Mix is diluted to 1x or 2x (with oligonucleotides and water), so we recommend doing this dilution before starting the run.

Depending on the volume lyophilised, the material of the vials (i.e., glass vs plastic) and the lyophiliser used, the cycle may be optimised.

If the cakes are not properly formed after the cycle, we recommend repeating with a more conservative cycle, in which the primary drying is carried at -50 °C and time is extended to guarantee the correct cake formation. Secondary drying phase could also be extended if required.

The primers should be designed to ensure that they have similar annealing temperatures, are specific to the target, and do not form primer dimers. The length of the reverse transcription phase can be extended to 10 minutes to ensure there is enough template for priming and amplification.