- Materials
-
- gDNA in 47 µl nuclease-free water
- DNA Control Sample (DCS)
- Consumables
-
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- OR 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- NEBNext FFPE DNA Repair Mix (NEB, cat # M6630)
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- Equipment
-
- 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)
- Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl, and tips
- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Thermal cycler
- Microfuge
- Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
- Vortex mixer
- Ice bucket with ice
- Pipetting troughs
-
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.
-
Prepare the DNA in nuclease-free water per sample:
- For R9.4.1 flow cells, transfer 1 μg (or 100-200 fmol) genomic DNA into a separate well of a 96-well plate or a 0.2 ml PCR tube strip
- For R10.3 flow cells, transfer 1.5-3 μg (or 150-300 fmol) genomic DNA into a separate well of a 96-well plate or a 0.2 ml PCR tube strip
- Adjust the volume to 47 μl with nuclease-free water
- Mix thoroughly by pipetting up and down, or by flicking the tube
- If necessary, seal and spin down briefly in an appropriate centrifuge
-
To each sample, add the following:
Between each addition, pipette mix 10-20 times.
Reagent Volume DNA CS 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 13 µl Total including DNA 60 µl -
Mix well by gently pipetting the entire volume within each well/tube up and down 10 times, or by flicking the tubes, and spin down.
-
Seal the plate, or close the tube lids.
-
Using a thermal cycler, incubate the samples at 20°C for 5 minutes and 65°C for 5 mins.
-
AMPure XP bead clean-up
It is recommended that the repaired/end-prepped DNA samples are subjected to the following clean-up with AMPure XP beads. This clean-up can be omitted for simplicity and to reduce library preparation time. However, it has been observed that omission of this clean-up can: reduce subsequent adapter ligation efficiency, increase the prevalence of chimeric reads, and lead to an increase in pores being unavailable for sequencing. If omitting the clean-up step, proceed to the next section (“Adapter Ligation and Clean-Up”).
-
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.
-
Keep the DNA samples in their original wells/PCR tubes. Add 60 µl of resuspended AMPure XP beads to each sample and mix by pipetting at least 100 µl up and down ten times. Retain any unused beads.
-
Incubate for 5 minutes at room temperature.
-
Prepare fresh 70% ethanol in nuclease-free water and pour into a pipetting trough. Allow enough for 500 µl per sample, with an excess to allow for dead volume within the pipetting trough. After the bead washing steps, discard any unused ethanol.
-
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.
-
Keeping the plate/tube strip on the magnet, wash each pellet of beads with 200 µl of the freshly-prepared 70% ethanol without disturbing the pellets. Remove the 70% ethanol using a pipette and discard.
-
Repeat the previous step.
-
Seal the plate, or close the tube lids. Spin down and place the plate/tube strip back on the magnet. Pipette off any residual ethanol.
-
Pour nuclease-free water into a pipetting trough. Allow enough for 61 µl per sample, with an excess to allow for dead volume within the pipetting trough.
-
Remove the plate/tube strip from the magnetic rack and resuspend each pellet in 61 µl nuclease-free water from the pipetting trough. Pipette the entire volume up and down ten times).
-
Seal the plate or close the tube lids, and incubate for 2 minutes at room temperature.
-
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 min.
-
Remove and retain 61 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.
-
Quantify 1 µl of each eluted sample using a Qubit fluorometer.