FAQs

Picolitre volume RPA reactions have been shown to work in published literature http://dx.doi.org/10.1371/journal.pone.0153359 .Obviously, this is subject to having the mechanism to generate such a volume.

Some small amount of the phosphocreatine in E-mix can occasionally form a precipitate. This should not affect RPA reactions as there is a large excess of phosphocreatine in the E-mix. Vortex the tube to resuspend precipitate and spin down any remaining precipitate before using the supernatant.

The recombinase and ancillary protein present in the 20x Core Reaction Mix are known to sometimes reversibly co-precipitate in 50% glycerol when stored at low temperatures. Warming the 20x Core Reaction Mix to room temperature and mixing will return them into solution, with no loss of function.

Yes, kits are fine to be at ambient temperatures for 2 – 3 weeks and should still work well. If you are unsure, please run a control test and send us the results, and we will be able to confirm that this is the case. For long term storage we suggest storage at -20˚C.

Yes, there are published papers demonstrating RPA use with these nucleotides. See http://dx.doi.org/10.7150/thno.15250 and http://dx.doi.org/10.1186/s13148-015-0100-6

Maybe. If you have developed primers using a TwistAmp® Basic kit, you will also need to include a probe if you are going to use a TwistAmp® exo or nfo kit. This and other factors mean that an optimal primer combination for a gel-based approach vs a fluorescence or lateral flow approach may not be the same. In general we

Oligos, 2x Reaction Buffer, water, dNTPs and 10x E-mix can be combined and stored on ice for a minimum of hours, and over-night in many cases (any effects on performance will be assay dependent and should be assessed independently by users). Once the 20x Core Reaction Mix is added short term storage on ice (assay dependent again) should also be

Yes. The polymerase and exonuclease III in TwistAmp® exo reactions are in competition. As the reaction runs out of energy, the exonuclease III starts to dominate. This leads to faster probe cleavage and a jump in fluorescence signal.

If positive results are seen in no template controls it is commonly caused by contamination, particularly, if the positive control oligonucleotides provided in the kit display false positives when no template is added. Contamination can occur if you are pipetting high copies (10^6 /µl or above) of template in the same area as carrying out RPA testing. We strongly recommend

The recommended concentration of probe in the reaction is 120nM. However, some TwistAmp® reactions benefit from being used with slightly different amounts of probe. Testing different concentrations of probe (from 50nM to 150nM) will help to optimise the performance of probe based detection formats of a given assay.