-
Prepare third party reagents in accordance with manufacturer's instructions, and place on ice:
-
Thaw the native barcodes at room temperature. Use one barcode per sample. Individually mix the barcodes by pipetting, spin down, and place them on ice.
-
Select two unique barcodes for each pair of samples to be run together.
-
In clean 1.5 ml Eppendorf DNA LoBind tubes, add the reagents in the following order per sample:
Reagent |
Volume |
End-prepped DNA |
7.5 µl |
Native barcode |
2.5 µl |
Blunt/TA Ligase Master Mix |
10 µl |
Total |
20 µl |
-
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
-
Incubate for 20 minutes at room temperature.
-
Add 2 µl of EDTA to each tube and mix thoroughly by pipetting and spin down briefly.
-
Pool the barcoded samples in a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Resuspend the Agencourt AMPure XP beads by vortexing.
-
Add 16 µl of Agencourt AMPure XP beads to the pooled reaction, and mix by pipetting.
-
Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
-
Prepare 500 µl of fresh 70% ethanol in nuclease-free water.
-
Spin down the sample and pellet on a magnet for 5 minutes. Keep the tube on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.
-
Keep the tube on the magentic rack 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 magnetic rack. Pipette off any residual ethanol. Allow the pellet 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 35 µl nuclease-free water by gently flicking.
-
Incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
-
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
-
Remove and retain 35 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Quantify 1 µl of eluted sample using a Qubit fluorometer.
-
End of step
Take forward the barcoded DNA library to the adapter ligation and clean-up step. However, you may store the sample at 4°C overnight.