- Materials
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- 1 μg or 100-200 fmol DNA
- Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)
- Consumables
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- NEBNext FFPE DNA Repair Mix (NEB, M6630)
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Equipment
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- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Microfuge
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Ice bucket with ice
- Vortex mixer
- Thermal cycler
- Qubit fluorometer (or equivalent for QC check)
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Day 1: Duplex experiment overview
In this step, the library for the Duplex experiment is prepared for sequencing, as follows: the extracted DNA is repaired and the ends prepared for adapter attachment using the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair/dA-tailing Module reagents. The sequencing adapters are attached to the DNA fragment ends before a clean-up step in preparation for sequencing on the PromethION Flow Cell.
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Thaw DNA Control Sample (DCS) at room temperature, spin down, mix by pipetting, and place on ice.
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Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.
For optimal performance, NEB recommend the following:
Thaw all reagents on ice.
Flick and/or invert the reagent tubes to ensure they are well mixed.
Note: Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.Always spin down tubes before opening for the first time each day.
The Ultra II End Prep Buffer and FFPE DNA Repair Buffer 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 30 seconds to solubilise any precipitate.
Note: It is important the buffers are mixed well by vortexing.The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.
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Prepare the DNA in nuclease-free water:
- Transfer 1 μg (or 100-200 fmol) input DNA into a 1.5 ml Eppendorf DNA LoBind tube.
- Adjust the volume to 47 μl with nuclease-free water.
- Mix thoroughly by pipetting up and down, or by flicking the tube.
- Spin down briefly in a microfuge
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In a 0.2 ml thin-walled PCR tube, mix the following:
Between each addition, pipette mix 10-20 times.
Reagent Volume DNA from the previous step 47 µl DNA CS (optional) 1 µl NEBNext FFPE DNA Repair Buffer 3.5 µl NEBNext FFPE DNA Repair Mix 2 µl Ultra II End-prep Reaction Buffer 3.5 µl Ultra II End-prep Enzyme Mix 3 µl Total 60 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
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Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
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Resuspend the AMPure XP Beads by vortexing.
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Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Add 60 µl of resuspended the AMPure XP Beads to the end-prep reaction and mix by flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 500 μl of fresh 80% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol 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 ethanol. 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 in 61 µl nuclease-free water. Incubate for 2 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 61 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.
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Optional actionIf a pause is required, the sample can be stored overnight at 4°C.
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Spin down the Ligation Adapter (LA) 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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Thaw the 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 DNA LoBind tube, mix in the following order:
Between each addition, pipette mix 10-20 times.
Reagent Volume DNA sample from the previous step 60 µl Ligation Buffer (LNB) 25 µl NEBNext Quick T4 DNA Ligase 10 µl Ligation Adapter (LA) 5 µl Total 100 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning 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 40 µl of resuspended AMPure XP Beads to the reaction and mix by flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
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Wash the beads by adding 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. 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 in 25 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 25 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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Make up your library to 32 µl at 10-20 fmol, using Elution Buffer (EB).