-
Mix the following reagents in a separate 0.2 ml thin-walled PCR tube for each pool of samples:
Reagent |
Volume |
Pool of barcoded DNA samples |
20 µl |
Ultra II End-prep reaction buffer |
7 µl |
Ultra II End-prep enzyme mix |
3 µl |
Nuclease-free water |
30 µl |
Total |
60 µl |
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Mix by pipetting.
-
Using a thermal cycler, incubate at 20° C for 15 minutes and 65° C for 5 mins.
-
Transfer each sample to a separate 1.5 ml Eppendorf DNA LoBind tube.
-
Resuspend the AMPure XP beads by vortexing.
-
Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.
-
Allow DNA to bind to beads for 5 minutes at room temperature.
-
Prepare sufficient fresh 70% ethanol in nuclease-free water.
-
Keep the tube on the magnet and wash the beads with 180 µl of freshly-prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
-
Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
-
Repeat the previous step.
-
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. 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 the pellet in 23.5 µl nuclease-free water. Incubate for 2 minutes at room temperature.
-
Pellet the beads on a magnet until the eluate is clear and colourless.
-
Remove and retain 23.5 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Quantify 1 µl of end-prepped DNA using a Qubit fluorometer.
-
End of step
Take forward 100–200 fmol of end-prepped DNA in 22.5 µl into native barcode ligation.