RPA1) Primers must be 30-35 bases2) Works best at constanttemperature (40°C)3) Amplicons of 80-400bp arepreferred4) TwistAmp? exo Probe required - see overleafSet-up (single-plex)11) Prepare reaction mix in 1.5ml tube: Primer A (10μM) 2.1 μl Primer B (10μM) 2.1 μl TwistAmp? exo Probe (10μM) 0.6 μl Rehydration Buffer 29.5 μl Template, RNase Inhibitor and dH2O 13.2 μl (Total Volume 47.5 μl)2) Add reaction mix to freeze-driedreaction. Pipette to mix.3) Add 2.5 μl of 280mM MgAc (supplied)and mix well to start reaction.4) Place strip in Twista? and start run: 40°C, 20 minutes.5) Remove strip after 5-7 minutes, invert vigorously 8-10 times to mix & spinbriefly,replace in Twista.WARNING: RPA REACTIONS START AT ROOM TEMPERATURE AS SOON AS MAGNESIUM IS ADDED.