- Materials
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- Long Fragment Buffer (LFB)
- Short Fragment Buffer (SFB)
- Elution Buffer (EB)
- Adapter Mix II (AMII)
- Consumables
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- NEBNext® Quick Ligation Module (NEB, E6056)
- NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
- 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) 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 T4 Ligase and the Adapter Mix II (AMII), 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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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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Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition.
Reagent Volume 100–200 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 -
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 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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Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl 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.
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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. 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 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 adapter ligated DNA using a Qubit fluorometer - recovery aim 50–100 fmol.
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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.