Clean Up Fragments

•

RSB (Resuspension Buffer)

•

Agencourt AMPure XP

•

Freshly prepared 80% ethanol (EtOH)

•

96-well PCR plate, semiskirted

•

Microseal 'B' adhesive film

1.

Vortex AMPure XP to resuspend.

2.

Add 34 µl AMPure XP to each well.

3.

Mix using either method:

•

Seal and shake at 2000 rpm for 1 minute, and then centrifuge at 280 × g for 10 seconds.

•

Slowly pipette until the beads are resuspended.

4.

Incubate at room temperature for 5 minutes.

5.

Place on the magnetic stand and wait 5 minutes.

6.

Remove and discard 67 µl supernatant.

7.

Wash beads as follows.

a.

Keep on the magnetic stand and add 175 µl fresh 80% EtOH to each well.

b.

Wait 30 seconds.

c.

Remove and discard all supernatant.

8.

Wash beads a second time.

9.

With a 20 µl pipette, remove all residual EtOH.

10.

Air-dry on the magnetic stand for 2 minutes. Do not overdry the beads.

11.

Remove from the magnetic stand.

12.

Add 22 µl RSB to each well.

13.

Mix using either method:

•

Seal and shake at 2200 rpm for 1 minute.

•

Slowly pipette until the beads are resuspended, and then seal.

14.

If shaking did not fully resuspend the beads, slowly pipette until the beads are resuspended, and then seal.

15.

Incubate at room temperature for 2 minutes.

16.

Centrifuge at 280 × g for 10 seconds.

17.

Place on the magnetic stand and wait 2 minutes.

18.

Transfer 20 µl supernatant to the corresponding well of a new PCR plate. Amplification PCR cycles vary by input amount. When multiple samples are prepared, transfer to separate plates according to the number of PCR cycles specified in the Amplify Library procedure.