- Materials
-
- AMPure XP Beads (AXP)
- Native Barcodes (NB01-24)
- EDTA (EDTA)
- Consumables
-
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 70% ethanol in nuclease-free water
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Equipment
-
- Magnetic rack
- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Microfuge
- Thermal cycler
- Ice bucket with ice
- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Optional equipment
-
- Qubit fluorometer (or equivalent for QC check)
-
Prepare the NEB Blunt/TA Ligase Master Mix according to the manufacturer's instructions, and place on ice:
Thaw the reagents at room temperature.
Spin down the reagent tubes for 5 seconds.
Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
-
Thaw the EDTA at room temperature and mix by vortexing. Then spin down 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 a unique barcode for each sample to be run together on the same flow cell. Up to 24 samples can be barcoded and combined in one experiment.
Please note: Only use one barcode per sample.
-
In clean 0.2 ml thin-walled PCR tubes, add the reagents in the following order per sample:
Between each addition, pipette mix 10 - 20 times.
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.
We expect ~20 µl per sample.
X6 samples X12 samples X24 samples Total volume 120 µl 240 µl 480 µl -
Resuspend the AMPure XP Beads (AXP) by vortexing.
-
Add AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting for a 0.4X clean.
Volume per sample x6 samples x12 samples x24 samples Volume of AXP 8 µl 48 µl 96 µl 192 µl -
Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
-
Prepare 2 ml 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 magnetic rack and wash the beads with 700 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.