Basic Information

RPA

1) Primers must be 30-35 bases

2) Works best at constant temperature (40-42°C)

3) Amplicons of 80-400bp are

Set-up (single-plex)1

1) 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 briefly

2) Add reaction mix to freeze-dried reaction. 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.