6.2. Purification of Amplified Indexed Library

The purpose of this step is to remove any residual primers and reagents from the pooled Amplified Indexed Libraries so that the preparations are ready for NGS.

1. Add 1.5x volume of SPRIselect magnetic beads (at room temperature) to pooled Amplified Indexed Libraries mix in an Eppendorf tube, and pipet up and down 5 times to thoroughly mix the bead suspension with the pooled Amplified Indexed Libraries.

2. Incubate the mixture for 5 minutes at room temperature.

3. Place the tube in the Magnetic Stand for 1 minute or until the solution is clear. Carefully remove and discard the supernatant without disturbing the pellet.

4. Add 500 µl of freshly prepared 80% ethanol to the tube.

5. Place the tube in the Magnetic Stand for 2 minutes or until the solution is clear. Carefully remove and discard the supernatant without disturbing the pellet.

6. Repeat steps 4 and 5 for a second wash.

7. Briefly centrifuge tubes at low speed and place the tubes in the Magnetic Stand. Use a 20-μl pipette to remove the residual ethanol droplets from the tube and air-dry the beads at room temperature for 2 minutes.

8. Add 45 µl of TE buffer to the pellet to disperse the beads and let stand for 1 minute.

9. Place the tube on the Magnetic Stand for 1 minute. Transfer 40 µl of the supernatant to a new Eppendorf tube.

10. Measure the concentration of pooled Amplified Indexed Libraries sample using the Qubit® dsDNA High Sensitivity Assay.

11. Dilute the pooled Amplified Indexed Libraries probe sample to 1.6 ng/µl, which corresponds to a concentration of 10 nM for the following NGS step (for NextSeq).

!Stopping Point in procedure: PCR products may be stored at 4°C overnight or at -20°C for a few days.