- Materials
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- Amplified DNA in 48 µl nuclease-free water
- AMPure XP Beads (AXP)
- Consumables
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- NEBNext® FFPE DNA Repair Mix (M6630) from the NEBNext® Companion Module v2 (NEB, E7672S or E7672L)
- NEBNext® Ultra II End Prep Enzyme Mix (E7646) from the NEBNext® Companion Module v2 (NEB, E7672S or E7672L)
- NEBNext® FFPE DNA Repair Buffer v2 (E7363) from the NEBNext® Companion Module v2 (NEB, E7672S or E7672L)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Microfuge
- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Ice bucket with ice
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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Check your flow cell.
We recommend performing a flow cell check before starting your library prep to ensure you have a flow cell with enough pores for a good sequencing run.
See the flow cell check instructions in the MinKNOW protocol for more information.
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Prepare the NEB 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.
Vortex the FFPE DNA Repair Buffer v2, or the NEBNext FFPE DNA Repair Buffer and Ultra II End Prep Reaction Buffer to ensure they are well mixed.
Note: These buffers may contain a white precipitate. If this occurs, allow the mixture(s) to come to room temperature and pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix.The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.
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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 Amplified DNA 48 µl NEBNext FFPE DNA Repair Buffer v2 7 µl NEBNext FFPE DNA Repair Mix 2 µl Ultra II End-prep Enzyme Mix 3 µl Total 60 µl If using the previous version of the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7180S or E7180L):
Between each addition, pipette mix 10-20 times.
Reagent Volume Amplified DNA 48 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 (AXP) 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 (AXP) 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.