RPA1) Primers must be 30-35 bases2) Works best at constanttemperature (40-42°C)3) Amplicons of 80-400bp areSet-up (single-plex)11) Prepare reaction mix in 1.5ml tube: Primer A (10μM) 2.4 μl Primer B (10μM) 2.4 μl Rehydration Buffer 29.5 μl Template, RNase Inhibitor and dH2O 13.2 μl (Total Volume 47.5 μl) Vortex and spin briefly2) 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) Incubate at 40-42°C for 20-40 minutes.4b) For low template copy number, remove strip after 5-7 minutes, invert vigorously 8-10 times to mix & spin briefly, replace in heating device.5) After 20-40 minutes, clean amplicons before running on agarose gels.WARNING: RPA REACTIONS START AT ROOM TEMPERATURE AS SOON AS MAGNESIUM IS ADDED.