Air-Dryable Probe Mix and Probe 1-Step Mix

The drying conditions provided in the user guide are merely an example of cycles tested in our laboratory. When air drying, it is important to achieve a Loss on Drying (LoD) of about 70-75%, which results in a dry gel containing only 5-10% water.

Drying times may vary depending on equipment used. When using this product for the first time with a specific oven, we recommend measuring LoD using several tubes at regular time intervals (e.g., after 60, 70, 80, 90 minutes) to determine the best protocol and achieve the LoD of 74.5 ± 0.5% or 70 ± 1% based on the mix (with or without oligos) and drying equipment used.

Temperatures can also be adjusted, but lower temperatures will require longer times, while higher temperatures risk affecting the integrity of the mix components, so we recommend using 40°C to minimize the possibility of encountering unexpected issues.

Although we have successfully lyophilized this mix, we do not recommend lyophilization. The excipient content is optimized for air drying, which significantly reduces the stability of the lyophilized product. We recommend using this mix solely for air drying and suggest using our Lyo-Ready product line for lyophilization protocols.

Absolutely. This is our first universal All-in-One mix, meaning it already contains RTase. Importantly, this does not interfere with the performance of our HS Taq polymerase, so it can be used with both RNA and DNA as the templates. We do not recommend simultaneous detection of RNA and DNA targets in a single multiplex reaction.

If 5–6 μL of mix (plus oligos) has been air dried per well, simply dissolve the gel with 20 μL of sample in water. Once resuspended, we recommend shaking the sealed plate for about 1 minute to achieve homogenous solution per well.

After drying, seal the tubes/plates. This way, they can be stored at room temperature for at least 1.5 years. For extended storage, we recommend storing plates in resealable bags with silica gel packets.

The dry mix behaves similarly to the wet mix, with no significant reduction in fluorescence or delay in amplification. It also demonstrates the same sensitivity and efficiency as all our 1-Step Probe Mixes, detecting up to 4 copies of a viral genome.

We observe no significant loss of activity (i.e., no reduction in fluorescence and delay of less than 1 Ct in amplification) after incubating the dry gel at 37°C for 3 months.

We have successfully amplified 2 separate sets of 4 targets concurrently. External users have reported successfully conducting more complex multiplex reactions. Please share any feedback and results on this with us at technical@pcrbio.com.

Upon receipt, the product should be stored between -30°C and -15°C. If stored correctly, the product will retain full activity for 12 months.

Please check that the equipment used for drying the mix is properly set up, connected to the power supply, and operating correctly.

Also make sure not to exceed the maximum of 5 – 6 μL of mix (including oligos and ROX dye) per well during air drying. Please ensure you are following the instructions provided in the guide. Optimal drying times may vary between different ovens. We recommend optimizing drying conditions for your reaction as needed, starting with the guidelines provided in our manual.

We recommend using the smallest volume possible to minimize protocol time. This means individually air drying 5 μL of the 4x mix (for a 20 μL reaction). If primers, probes, and optionally ROX (if required by the PCR machine) are added before executing the protocol, high concentration stock solutions should be prepared, to add the smallest possible volume to the 4x mix. We advise not to exceed 1 μL additional. Larger volumes will require longer drying times and higher LoD to achieve the same residual water percentage in the dry product. When air drying 6 μL of the mix with primers and probes, the LoD needs to achieve 74.5% ± 0.5%, and so we recommend air drying the mix at 40°C for 80-90 minutes.

We do not recommend drying more than 5 – 6 μL of mix (plus oligos) per well as directed in the guidelines provided. Multiple plates can be dried at once to increase production. We do not recommend air drying larger volumes and suggest using lyophilization, with our Lyo-Ready product line.