Double-AMPure purification protocol for 16S amplicon pools Before you begin

1. Set a heat block to 96°C. 2. Prepare 70% ethanol in the amount needed (400 μl per Amplicon). For 10 ml, add 7 ml of 100%

ethanol to 3 ml of Molecular Biology Grade Water, and vortex. Experimental procedure:

1. Briefly centrifuge the PCR tubes. 2. Pipet 100 μl of molecular biology grade water into 1.5 ml tubes (one tube per Amplicon pool). 3. Transfer 100 μl of each Amplicon pool from the PCR tubes to each 1.5 ml tube (current volume

200 μl). 4. Vortex the AMPure bead bottle for 20 seconds or until the beads are completely resuspended. 5. Add 120 μl of AMPure beads to each tube (final ratio 1 : 0.6 à 200 µl sample + 120 µl

AMPure beads)*, and mix thoroughly by vortexing for 5 sec. 6. Incubate the bead-DNA mixture for 10 min at 96°C on the heat block. 7. Incubate for 8 min at room temperature. Spin down (2 sec). 8. Place the tubes in a Magnetic Particle Collector (MPC) and incubate for 5 min at room

temperature. 9. With the tubes still on the MPC, carefully remove and discard the supernatant without disturbing

the beads. 10. Perform a quick spin and put the tubes back on the MPC. Remove the rest of supernatant

completely. 11. Add 100 μl of 1× TE to each tube. Vortex 5 sec or until the pellet is completely resuspended.

Spin down (2 sec). 12. Place the tubes in the MPC and incubate for 2 min at room temperature. 13. With the tubes still in the MPC, carefully transfer the supernatants (100 μl library) to a set of fresh

1.5 ml tubes. 14. Pipet 100 μl of Molecular Biology Grade water into each 1.5 ml tubes containing the library. 15. Repeat steps 4 to 10, then go to step 16. Set the heat block to 37°C. 16. Remove the tubes from the MPC and add 200 μl of 70% ethanol (freshly prepared) to each tube. 17. Vortex the tubes for 5 sec. The pellet may not resuspend completely; this is acceptable. Spin

down (2 sec). 18. Place the tubes on the MPC and incubate for 1 min. 19. With the tubes still on the MPC, carefully remove and discard the supernatant without disturbing

the beads. 20. Perform a quick spin and put the tubes back on the MPC. Remove the rest of supernatant

completely. 21. Repeat steps 16 – 20. Remove as much of the supernatant as possible. 22. Place the open tubes on a heat block set at 37°C until the pellet is completely dry (about 2 min).

Do not leave the tubes on the heat block longer than necessary to avoid over-drying (avoid visible cracks on AMPure bead pellet).

23. Remove the tubes from the heat block. 24. Add 10 μl of 1× TE to each tube. Vortex 5 sec or until the pellet is completely resuspended. Spin

down (2 sec). 25. Place the tubes in the MPC and incubate for 2 min at room temperature. 26. With the tubes still in the MPC, carefully transfer the supernatants to a set of fresh screw cap o-

ring 1.5 ml tubes. 27. Store the purified Amplicon pools individually at -20ºC until ready to proceed.

* The ratio to be used (Sample : AMPure beads) depends on the library size. Please see the TCB 2011-007 (version May 2013) to determine the appropriate cutoff.