-
Resuspend the AMPure XP beads by vortexing.
-
Add 40 µl of resuspended AMPure XP beads to the adapter ligation reaction from the previous step and mix by pipetting.
-
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
-
Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
-
Add 200 μl of Wash Buffer (WSB) to the beads. Resuspend the beads by pipetting up and down. Return the tube to the magnetic rack, allow beads to pellet and pipette off the supernatant.
-
Repeat the previous step.
-
Important
Agitating the beads results in a more efficient removal of free adapter, compared to adding the wash buffer and immediately aspirating.
-
Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
-
Remove the tube from the magnetic rack and resuspend pellet in 13 µl of Elution Buffer (EB).
-
Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
-
Pellet the beads on a magnet until the eluate is clear and colourless.
-
Remove and retain 13 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Quantify 1 µl of eluted cDNA using a Qubit fluorometer - recovery aim ~60 ng.
-
End of step
The prepared library is used for loading onto the flow cell. Store the library on ice until ready to load.