- Materials
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- Ligation Adapter (LA)
- Ligation Buffer (LNB) from the Ligation Sequencing Kit
- Long Fragment Buffer (LFB)
- Short Fragment Buffer (SFB)
- AMPure XP Beads (AXP)
- Elution Buffer (EB) from the Oxford Nanopore sequencing kit
- Consumables
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- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- NEBNext Quick Ligation Module (NEB, E6056)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Magnetic rack
- Microfuge
- Vortex mixer
- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
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Spin down the Ligation Adapter (LA) and Quick T4 Ligase, and place on ice.
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Thaw 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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Thaw either Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then 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 DNA sample from the previous step 30 µl Ligation Buffer (LNB) 12.5 µl NEBNext Quick T4 DNA Ligase 5 µl Ligation Adapter (LA) 2.5 µl Total 50 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
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Incubate the reaction for 10 minutes at room temperature.
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Resuspend the AMPure XP Beads (AXP) by vortexing.
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Add 20 µl of resuspended AMPure XP beads (AXP) to the reaction and mix by flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 5 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 either 125 μl Long Fragment Buffer (LFB) or 125 μl Short Fragment Buffer (SFB). Flick the beads to resuspend, 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 pellet in 7 µl Elution Buffer (EB). Incubate for 10 minutes at room temperature. For high molecular weight DNA, incubating at 37° C can improve the recovery of long fragments.
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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 7 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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Prepare your final library to 5-10 fmol in 5 µl of Elution Buffer (EB).