5.2. Purification of the Amplified Indexed Libraries

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 1.8x volume of Agencourt® AMPure® XP Reagent (at room temperature) to pooled Amplified Indexed Libraries and Negative Control sample 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 2 minutes at room temperature.

3. Place tube in the Thermo Fisher Dynabeads® MPC®-S 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, and wash the beads by pipetting.

5. 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.

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 25 μl of fresh PCR-Grade Water 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 20 μl of the supernatant to a new Eppendorf tube.

10. Measure the concentration of both pooled Amplified Indexed Libraries sample and Negative Control sample using the Qubit® dsDNA High Sensitivity Assay. Subtract the concentration reading (ng/μl) of the Negative Control sample from the pooled Amplified Indexed Libraries reading.

11. Dilute the pooled Amplified Indexed Libraries probe sample to 1.8 ng/μl, which corresponds to a concentration of 10nM for the following NGS step.