- Materials
-
- ≥6 ng of recovered human cfDNA per sample (12 samples)
- AMPure XP Beads (AXP)
- Consumables
-
- NEBNext FFPE DNA Repair v2 Module (NEB, E7360)
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- 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
-
- 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
-
- Qubit fluorometer (or equivalent for QC check)
-
Prepare the NEBNext FFPE DNA Repair Mix, the NEBNext FFPE DNA Repair Buffer v2 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, NEB Thermoliable Proteinase K or Ultra II End Prep Enzyme Mix.Always spin down tubes before opening for the first time each day.
The FFPE DNA Repair Buffer v2 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 v2 may have a yellow tinge and is fine to use if yellow.
-
Use ≥6 ng of recovered cfDNA, per sample as input. Inputs should be normalised so the same mass of sample is used in each reaction.
-
Prepare your cfDNA samples in nuclease-free water:
For each sample, ensure you have ≥6 ng of extracted cfDNA from the sample extraction, and transfer this into a 0.2 ml thin-walled PCR tube.
Note: You should have 12 sample tubes.Adjust the volume to 20 μl with nuclease-free water.
Mix thoroughly by pipetting up and down, or by flicking the tube.
Spin down briefly in a microfuge.
-
In each of the 0.2 ml thin-walled PCR tube containing your cfDNA samples, mix the following:
Reagent Volume cfDNA from the previous step 20 µl NEBNext FFPE DNA Repair Buffer v2 3 µl NEBNext FFPE DNA Repair Mix v2 0.9 µl Total 23.9 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
-
Using a thermal cycler with a heated lid set to 50°C, incubate the reaction at 37°C for 15 minutes and hold at 4°C.
-
Remove the reaction from the thermal cycler and place the tube on ice.
-
Keeping the tubes on ice, add 0.9 µl of NEBNext Thermolabile Proteinase K directly to each of the repaired reaction mixtures.
-
Mix by pipetting 10 times, followed by spinning down quickly to collect all liquid from the sides of the tube.
-
Using a thermal cycler with a heated lid set to 75°C, incubate at 37°C for 15 minutes and 65°C for 5 minutes, then hold at 4°C.
-
Remove the reaction from the thermal cycler and place the tube on ice.
-
Keeping the tube on ice, add 1.3 µl of NEBNext Ultra II End Prep Enzyme Mix directly to the reaction mixture for a total volume of 26.1 µl.
-
Mix by pipetting 10 times, followed by spinning down quickly to collect all liquid from the sides of the tube.
-
Using a thermal cycler with a heated lid set to 75°C, incubate at 20°C for 30 minutes and 65°C for 30 minutes, then hold at 4°C.
-
Resuspend the AMPure XP Beads (AXP) by vortexing.
-
Transfer the each cfDNA sample into a separate clean 1.5 ml Eppendorf DNA LoBind tube.
-
Add 80 µl of resuspended the AMPure XP Beads (AXP) to each end-prep reaction and mix by flicking the tube.
-
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
-
Prepare 5 ml of fresh 80% ethanol in nuclease-free water.
Note: Ensure you prepare sufficient 80% ethanol for your 12 samples.
-
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.
-
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.
If the pellet was disturbed, wait for beads to pellet again before removing the ethanol.
-
Repeat the previous step.
-
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.
-
For each sample, remove the tube from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Incubate for 2 minutes at room temperature.
-
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
-
For each sample, remove and retain 10 µl of eluate into a separate clean 1.5 ml Eppendorf DNA LoBind tube.
Note: Ensure your samples are processed separately. At this stage they are not yet barcoded.
-
Quantify 1 µl of each eluted sample using a Qubit fluorometer.
Note: You should expect to recover approximately 75% of your input mass. For example, from 6 ng of cfDNA, a yield of approximately 4.5 ng is expected.