- Materials
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- Long Fragment Buffer (LFB)
- Elution Buffer (EB)
- Adapter Mix II T (AMII T)
- Consumables
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- NEBNext® Quick Ligation Module (NEB, E6056)
- NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
- 1.5 ml Eppendorf DNA LoBind tubes
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Microfuge
- Magnetic rack
- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Thermal cycler
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Ice bucket with ice
- Qubit fluorometer (or equivalent)
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Thaw the Elution Buffer (EB) and NEBNext Quick Ligation Reaction Buffer (5x) at room temperature, mix by vortexing, spin down and place on ice. Check the contents of each tube are clear of any precipitate.
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Spin down the Quick T4 Ligase and the Adapter Mix II T (AMII T), and place on ice.
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To enrich for DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at room temperature, mix by vortexing, spin down and place on ice.
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In a 1.5 ml Eppendorf LoBind tube, mix in the following order:
Reagent Volume Pooled barcoded sample 30 µl Adapter Mix II T (AMII T) 5 µl NEBNext Quick Ligation Reaction Buffer (5X) 10 µl Quick T4 DNA Ligase 5 µl Total 50 µl -
Ensure the components are thoroughly mixed by pipetting, and spin down.
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Incubate the reaction for 10 minutes at room temperature.
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Resuspend the Agencourt AMPure XP beads by vortexing.
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Add 20 µl of resuspended Agencourt AMPure XP beads to the reaction and mix by pipetting.
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Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
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Wash the beads by adding 125 μl Long Fragment Buffer (LFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
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Repeat the previous step.
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Spin down and place the tube back on the magnet. Pipette off any residual supernatant. 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 in 30 µl Elution Buffer (EB).
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Spin down and incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
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Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
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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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Optional actionQuantify 1 µl of eluted sample using a Qubit fluorometer.
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Optional actionIf quantities allow, the libraries may be diluted in Elution Buffer (EB) for splittling across multiple flow cells.