- Materials
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- Long Fragment Buffer (LFB)
- Short Fragment Buffer (SFB)
- Elution Buffer (EB)
- Native Adapter (NA)
- AMPure XP Beads (AXP)
- Consumables
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- NEBNext® Quick Ligation Module (NEB, E6056)
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Equipment
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- Microfuge
- Magnetic rack
- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Thermal cycler
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Ice bucket with ice
- Qubit fluorometer (or equivalent for QC check)
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Prepare the NEBNext Quick Ligation Reaction Module according to the manufacturer's instructions, and place on ice:
Thaw the reagents at room temperature.
Spin down the reagent tubes for 5 seconds.
Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
Note: Do NOT vortex the Quick T4 DNA Ligase.
The NEBNext Quick Ligation Reaction Buffer (5x) may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for several seconds to ensure the reagent is thoroughly mixed.
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Spin down the Native Adapter (NA) and Quick T4 DNA Ligase, pipette mix and place on ice.
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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 LoBind tube, mix in the following order:
Between each addition, pipette mix 10 - 20 times.
Reagent Volume Pooled barcoded libraries 30 µl Native Adapter (NA) 5 µl NEBNext Quick Ligation Reaction Buffer (5X) 10 µl Quick T4 DNA Ligase 5 µl Total 50 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
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Incubate the reaction for 20 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 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 on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
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Wash the beads by adding either 125 μl Long Fragment Buffer (LFB) or 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 pellet in 15 µl Elution Buffer (EB).
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Spin down and incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
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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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Depending on your DNA library fragment size, prepare your final library in 12 µl of Elution Buffer (EB).
Fragment library length Flow cell loading amount Very short (<1 kb) 100 fmol Short (1-10 kb) 35–50 fmol Long (>10 kb) 300 ng Note: If the library yields are below the input recommendations, load the entire library.
If required, we recommend using a mass to mol calculator such as the NEB calculator.
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Optional actionIf quantities allow, the library may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.
Depending on how many flow cells the library will be split across, more Elution Buffer (EB) than what is supplied in the kit will be required.