- Materials
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- Adapter Mix (AMX)
- Ligation Buffer (LNB)
- Short Fragment Buffer (SFB)
- Elution Buffer (EB)
- Consumables
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- NEBNext Quick Ligation Module (NEB, E6056)
- 1.5 ml Eppendorf DNA LoBind tubes
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Equipment
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- Magnetic rack suitable for 0.2 ml PCR tubes
- Microfuge
- Vortex mixer
- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
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Spin down the Adapter Mix (AMX) and Quick T4 Ligase, and place on ice.
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Thaw the Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
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Thaw the Elution Buffer (EB) at room temperature and mix by vortexing. Then spin down and place on ice.
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To retain DNA fragments of all sizes, thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.
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In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:
Reagent Volume End-prepped DNA sample 60 µl Adapter Mix (AMX) 5 µl Ligation Buffer (LNB) 25 µl NEBNext Quick T4 DNA Ligase 10 µl Total 100 µl -
Mix gently by flicking the tube and spin down.
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Incubate the reaction for 10 minutes at room temperature.
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Resuspend the AMPure XP beads by vortexing.
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Add 40 µl of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
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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 a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
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Wash the beads by adding 200 µl of Short Fragment Buffer (SFB). 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 15 µl Elution Buffer (EB). Spin down and incubate for 10 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear.
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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.
Dispose of the pelleted beads.
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Quantify 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 splitting across multiple flow cells.
Additional buffer for doing this can be found in the Sequencing Auxiliary Vials expansion (EXP-AUX001), available to purchase separately. This expansion also contains additional vials of Sequencing Buffer (SQB) and Loading Beads (LB), required for loading the libraries onto flow cells.