FAQs

Yes. Biotinylated primers or ones with fluorophore should work the same as unmodified primers in an RPA reaction. We have yet to see any differences.

When performing a primer screen, one does not normally need to use specially purified oligonucleotides. However, as with use in other techniques, we have noted batch-to-batch variations in the quality of primer preparations. For applications where consistency is critical, we would therefore recommend the use of more purified primers once good primers are identified.

Yes. The crowding agent and proteins in RPA reactions can interfere with the antibodies on a lateral flow strip, so you can get nonspecific binding and false positive signals if you do not dilute them sufficiently.

Yes. The crowding agent and proteins in an RPA reaction interfere with normal agarose gel electrophoresis, so you will probably get a smear, rather than a clean band if you do not clean up your reaction.

No. Any detection method can be used to evaluate and compare the performance of potential primer pairs. However, real-time monitoring of the amplification with a detection probe has proven to be the fastest and least laborious method for screening high sensitivity primers.

No. For TwistAmp® Basic and nfo kits, any device that can hold a steady temperature of 37-42°C is fine. For real-time reactions using TwistAmp® exo kits, any plate reader or real-time thermal cycler that can excite and detect the fluorophores you are using, and hold a steady temperature of 37-42°C is fine. NB heated lids should be switched off when

Yes. The onset time of detectable amplification for a given assay will depend on the amount of starting template material – the more template copies there are to start with, the earlier the detection time will be. However, exploiting this ‘time-based’ quantification demands a careful experimental setup ensuring simultaneous initiation of compared reactions (e.g. through ‘Magnesium start’). The resolution of

The polymerase used in TwistAmp® Basic reactions does not have an editing function, so this should be possible. However, we have not tested this ourselves.

No. Most popular PCR probe systems are not suited for use with the TwistAmp® process. In particular those systems employing the 5’ to 3’ nuclease activity of polymerases cannot be used with the TwistAmp® system as such enzymatic activity is fundamentally incompatible with the RPA biochemistry.

Yes. It is possible to use intercalating dyes to quantify and monitor the TwistAmp® reactions in progress. However, as with PCR, the dye typically binds to any double-stranded DNA, so primer noise can generate false positive signals. This noise is made worse as the dye is monitored at a low temperature – in PCR assays the temperature is often such