Reliable amplification for various targets has been achieved using 40 cycles with 1 s denaturation and 3 s annealing and extension (2-step cycling). Successful amplification under these conditions requires reasonably short amplicons and should be tested on case-by-case basis for all new targets added to an experiment.

Results will differ depending on the plant species and tissue type. Our inhibitor-tolerant chemistry shows moderate resistance to chlorophyll and polyphenols, such as catechin and quercetin, but low resistance to tannic acid, cellulose and pectin. Thus, testing the mix on the desired sample types is recommend. All plant tissues will require mechanical lysis in an appropriate extraction buffer to achieve disruption of cell walls and membranes, prior to use with inhibitor-tolerant mixes. We recommend testing a small number of reactions on the target sample type. Please reach out to our team [email protected] to request a product sample, or if you need assistance in planning such test reactions.

Saliva samples do not require pre-treatment. However, dilution in appropriate sample transport medium is recommended. We highly recommend the use of UTM which don’t contain guanidinum chloride.

We have successfully carried out multiplex reactions on up to 5 μL of blood sample (1.25 μL of crude blood diluted 1:4 in a NaCl/EDTA solution) per 20 μL reaction. This does lead to a reduction in overall fluorescence but does not inhibit qPCR reactions themselves, nor does it limit the sensitivity of the mix.

We have successfully tested up to 10% crude saliva per reaction, with best, most-consistent results when using 2.5% saliva

Yes. While the mix is inhibitor-tolerant, it is not completely resistant to the presence of inhibitors. Different inhibitors and higher concentrations can still inhibit qPCR reactions. Given the sensitivity of the mix, if a significant delay is observed, we recommend diluting the sample before using in qPCR reactions.

Yes, this is expected. Crude biological samples, particularly saliva, contain different levels of PCR inhibitors. We have observed a range of 0.5-3.5 cycles delay in amplification of the same target with different saliva samples. The same crude biological sample may also show different levels of inhibition over time.

All our inhibitor-tolerant mixes have been tested on and tolerate crude saliva and crude blood. Additionally, they have been extensively tested in the presence of multiple PCR inhibitors found in clinical, plant, environmental, and laboratory chemicals . These include: hemin, haemoglobin, heparin, lactoferrin, IgG immunoglobulins, guanidine, SDS, ethanol, urea, sodium citrate, humic acid, cellulose, pectin, quercetin, catechin, tannic acid, and chlorophyll B. The extent of tolerance varies by compound. The mixes may also be tolerant to other compounds, not listed, or not yet tested.