- Materials
-
- Strand-switched cDNA in 20 µl
- AMPure XP Beads (AXP)
- Consumables
-
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Equipment
-
- P1000 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- Thermal cycler
- Microfuge
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Ice bucket with ice
- Optional equipment
-
- Qubit fluorometer (or equivalent for QC check)
-
Prepare the NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer's instructions, and place on ice:
For optimal perfomance, 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 Ultra II End Prep Enzyme Mix.Always spin down tubes before opening for the first time each day.
The Ultra II End Prep 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.
-
Combine the following reagents in a 0.2 ml PCR tube:
Reagent Volume cDNA sample 20 µl Nuclease-free water 30 µl Ultra II End-prep reaction buffer 7 µl Ultra II End-prep enzyme mix 3 µl Total 60 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
-
Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
-
Resuspend the AMPure XP Beads (AXP) by vortexing.
-
Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Add 60 µl of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
-
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
-
Prepare 500 μl of fresh 80% ethanol in nuclease-free water.
-
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.
-
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.
-
Remove the tube from the magnetic rack and resuspend pellet in 61 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.
-
Remove and retain 61 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Quantify 1 µl of eluted sample using a Qubit fluorometer.