- Materials
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- Elution Buffer (EB)
- Short Fragment Buffer (SFB)
- Adapter Mix II (AMII)
- Consumables
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- NEBNext Quick Ligation Module (NEB, E6056)
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Microfuge
- Magnetic rack
- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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Adapter Mix II Expansion use
Protocols that use the Native Barcoding Expansions require 5 μl of AMII per reaction. Native Barcoding Expansions EXP-NBD104/NBD114 and EXP-NBD196 contain sufficient AMII for 6 and 12 reactions, respectively (or 12 and 24 reactions when sequencing on Flongle). This assumes that all barcodes are used in one sequencing run.
The Adapter Mix II expansion provides additional AMII for customers who are running subsets of barcodes, and allows a further 12 reactions (24 on Flongle).
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Thaw the Elution Buffer (EB), Short Fragment Buffer (SFB), and NEBNext Quick Ligation Reaction Buffer (5x) at room temperature and mix by vortexing. Then spin down and place on ice. Check the contents of each tube are clear of any precipitate.
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Spin down the T4 Ligase and the Adapter Mix II (AMII), and place on ice.
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Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition.
Reagent Volume Pooled barcoded sample (30-50 ng) 30 µl Adapter Mix II (AMII) 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 AMPure XP beads by vortexing.
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Add 20 µl of resuspended 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 the beads on a magnet for 5 minutes. Keep the tube on the magnet until the eluate is clear and colourless, and pipette off the supernatant.
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Wash the beads by adding 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. Keep the tube on the magnet until the eluate is clear and colourless. 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 by pipetting in 15 µl Elution Buffer (EB). Spin down and incubate for 5 minutes at room temperature.
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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 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 library 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.