Basic Information:RPA1) Primers must be 30-35 bases2) Works best at constant temperature (37-39°C)3) Amplicons of 80-400bp are preferred4) TwistAmp? LF Probe or TwistAmp?exo Probe 1 required - see overleafSet-up (single-plex)21) Prepare reaction mix in 1.5ml tube: Primer A (10μM) 2.1 μl Primer B (10μM) 2.1 μl TwistAmp? LF Probe (10μM) 0.6 μl Rehydration Buffer 29.5 μl Template 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 37-39°C for 20-40 minutes.4b) For low template copy number,remove strip after 4 minutes, vortex &spin briefly, replace in heating device. 5) For analysis by lateral flow, dilute reacion products 1/50 with PBST and load 10 μl onto the sample pad of appropriate strip (eg Milenia? HybriDetect strips).6) Place strip in PBST running buffer.WARNING: RPA REACTIONS START AT ROOM TEMPERATURE AS SOON AS MAGNESIUM IS ADDED.
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