- Materials
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- Adapter Mix H (AMX H)
- Ligation Buffer (LNB)
- Long Fragment Buffer (LFB)
- Short Fragment Buffer (SFB)
- Elution Buffer (EB)
- 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
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Equipment
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- Magnetic rack suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, cat # 12027)
- OR magnetic separator suitable for 0.2 ml PCR tube strips, e.g. DynaMag™-PCR Magnet (Thermo Fisher, #492025) or DynaMag™-96 Side Magnet (Thermo Fisher, #12331D)
- Microfuge
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Vortex mixer
- Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl, and tips
- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Pipetting troughs
- Qubit fluorometer (or equivalent for QC check)
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Spin down the Adapter Mix H (AMX H) 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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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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To each repaired/end-prepped DNA sample (60 µl), add the following:
Between each addition, pipette mix 10 - 20 times.
Reagent Volume Ligation Buffer (LNB) 25 µl NEBNext Quick T4 DNA Ligase 10 µl Adapter Mix H (AMX H) 5 µl Total 40 µl Total including DNA 100 µl -
Mix well by gently pipetting the entire volume within each well/tube up and down 10 times.
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Incubate the reaction for 10 minutes at room temperature.
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Resuspend the AMPure XP beads by vortexing and transfer to a pipetting trough. Ensure that the volume transferred is enough for 60 µl to be added to each DNA sample, with an excess to allow for dead volume within the pipetting trough.
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Add 40 µl of resuspended AMPure XP beads to each sample and mix by pipetting the entire combined volume up and down 10 times.
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Incubate for 5 minutes at room temperature.
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Add sufficient Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) to a pipetting trough. Allow enough for 400 µl per sample, with an excess to allow for dead volume within the pipetting trough. Retain any unused reagent after the wash steps.
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Pellet the beads on a magnet for at least 2 minutes, or until the supernatant is clear. Keep the plate/tube strip on the magnet and pipette off the supernatant.
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Remove the plate/tube strip from the magnetic rack and wash each pellet of beads by adding either 200 μl Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB). Resuspend each pellet thoroughly by pipetting the entire volume of buffer up and down ten times. Fully resuspending the beads at this step ensures optimal kit performance. Return the plate/tube strip to the magnetic rack and allow the beads to pellet until the supernatant is clear. Remove the supernatant using a pipette and discard.
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Repeat the previous step.
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Seal the plate, or close the tube lids. Spin down and place the plate/tube strip back on the magnet. Pipette off any residual supernatant.
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Add sufficient Elution Buffer (EB) to a pipetting trough. Allow enough for 15 µl per sample, with an excess to allow for dead volume within the pipetting trough. Retain any unused Elution Buffer (EB) after the elution step.
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Remove the plate/tube strip from the magnetic rack and resuspend each pellet in 15 µl Elution Buffer (EB) from the pipetting trough, pipetting the entire volume up and down 10 times.
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Seal the plate (or close the tube lids), and incubate for 10 minutes at 37°C in a thermal cycler. Any heated lid used should be limited to 50°C.
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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 each eluate in a separate, clean well/tube within a 96-well PCR plate or PCR tube strip. 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-AUX002), available to purchase separately. This expansion also contains additional vials of Sequencing Buffer II (SBII) and Loading Beads II (LBII), required for loading the libraries onto flow cells.