- Materials
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- AMPure XP Beads (AXP)
- Elution Buffer from the Oxford Nanopore kit (EB)
- Rapid Adapter (RA)
- Adapter Buffer (ADB)
- Consumables
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- Freshly prepared 80% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- 5 ml Eppendorf DNA LoBind tubes
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Microfuge
- Centrifuge capable of taking 96-well plates
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Ice bucket with ice
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Qubit fluorometer plate reader (or equivalent for QC check)
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Briefly spin down the Barcode Attachment Plate to collect the liquid at the bottom of the wells prior to opening.
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Pool the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.
We expect to have about ~10 µl per sample.
X24 samples X48 samples X96 samples Total volume ~240 µl ~480 µl ~960 µl -
Mix pooled samples by vortexing.
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Transfer half of the barcoded pooled sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Per sample, we expect to take forward ~5 µl.
X24 samples X48 samples X96 samples Example volume 120 µl 240 µl 480 µl -
Resuspend the AMPure XP Beads (AXP) by vortexing.
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To the pooled barcoded sample, add an equal volume of resuspended AMPure XP Beads (AXP, or SPRI) and mix by pipetting.
Example volume X24 samples X48 samples X96 samples Volume of 1X AXP 120 µl 240 µl 480 µl -
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare at least 3 ml of fresh 80% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
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Keep the tube on the magnet and wash the beads with 1 ml of freshly-prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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Briefly 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.
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Remove the tube from the magnetic rack and resuspend the pellet by pipetting in 15 µl Elution Buffer (EB). Incubate for 10 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify DNA concentration by using the Qubit dsDNA HS Assay Kit.
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Take forward 11 µl of your eluted DNA library.
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In a fresh 1.5 ml Eppendorf DNA LoBind tube, dilute the Rapid Adapter (RA) as follows and pipette mix:
Reagent Volume Rapid Adapter (RA) 1.5 μl Adapter Buffer (ADB) 3.5 μl Total 5 μl -
Add 1 µl of the diluted Rapid Adapter (RA) to the barcoded DNA.
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Mix gently by flicking the tubes, and spin down.
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Incubate the reaction for 5 minutes at room temperature.