- Materials
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- Wash Buffer (WSB)
- Elution Buffer (EB)
- Adapter Mix II (AMII)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- NEBNext Quick Ligation Module (NEB, E6056)
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Equipment
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- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Vortex mixer
- Microfuge
- Ice bucket with ice
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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 Wash Buffer (WSB), Elution Buffer (EB) and NEBNext Quick Ligation Reaction Buffer (5x) at room temperature and mix by vortexing. Then spin down and place on ice. Check the contents or 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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Check the contents of each tube are clear of any precipitate and are thoroughly mixed before setting up the reaction.
- Check that there is no precipitate present (DTT in the Blunt/TA Master Mix, if used, can sometimes form a precipitate)
- Spin down briefly before accurately pipetting the contents into the reaction
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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 200 fmol pooled barcoded sample 65 µl Adapter Mix II (AMII) 5 µl NEBNext Quick Ligation Reaction Buffer (5X) 20 µl 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 50 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 5 minutes at room temperature.
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Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
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Add 140 μl of the Wash Buffer (WSB) to the beads. Close the tube lid, and resuspend the beads by flicking the tube. Return the tube to the magnetic rack, allow beads to pellet and pipette off the supernatant.
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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 13 µl of Elution Buffer (EB).
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Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Pellet the beads on the magnet until the eluate is clear and colourless.
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Remove and retain 13 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of eluted sample using a Qubit fluorometer.