- Materials
-
- cDNA Primer (cPRM)
- Elution Buffer from the Oxford Nanopore kit (EB)
- Consumables
-
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533S)
- Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
-
- Thermal cycler
- Vortex mixer
- Hula mixer (rotator mixer)
- Ice bucket with ice
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
-
In a 0.2 ml thin-walled PCR tube, prepare the following PCR reaction:
Reagent Volume cPRM 1 μl Nuclease-free water 4 μl LongAmp Hot Start Taq 2X Master Mix 25 μl Total 30 μl -
Resuspend the amplicon-bead conjugate by pipetting and then transfer 20 μl of the conjugate into the 0.2 ml thin-walled PCR tube containing the PCR reaction. Mix by pipetting.
-
Do not spin down the tube; transfer immediately to the thermal cycler and amplify using the following cycling conditions:
Cycle step Temperature Time No. of cycles Initial denaturation 94°C 3 min 1 Denaturation
Annealing
Extension94°C
62°C
65°C15 s
15 s
6 min
4Final extension 65°C 10 min 1 Hold 4°C ∞ - -
Resuspend the AMPure XP beads by vortexing.
-
Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Add 40 µl of resuspended AMPure XP beads to the 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 70% 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 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
-
Repeat the previous step.
-
Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual ethanol. Allow to dry for 30 seconds, but do not dry the pellets to the point of cracking.
-
Remove the tube from the magnetic rack and resuspend the pellet in 15 µl Elution Buffer (EB).
-
Pellet the beads on the magnet until the eluate is clear and colourless.
-
Remove and retain 15 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads
-
Quantify 1 µl of eluted sample using a Qubit fluorometer - recovery aim >50 ng total.