- Materials
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- AMPure XP Beads (AXP, or SPRI)
- Elution Buffer (EB)
- Rapid Adapter F (RAP F)
- Consumables
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- Freshly prepared 80% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- 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
- Optional equipment
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- 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 -
Resuspend the AMPure XP Beads (AXP, or SPRI) by vortexing.
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To the entire pooled barcoded sample, add an equal volume of resuspended AMPure XP Beads (AXP, or SPRI) and mix by flicking the tube.
x24 samples x48 samples x96 samples Volume of AXP to add 240 µl 480 µl 960 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 1.5 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 30 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 30 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads
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Quantify DNA concentration by using the Qubit dsDNA HS Assay Kit.
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Take forward 600–800 ng of library and make up the volume to 11 μl with EB.
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Add 1 µl of Rapid Adapter F (RAP F) to 11 µl of barcoded DNA.
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Incubate at room temperature for 5 minutes.