- Materials
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- 20 μl of the amplicon-bead conjugate
- PCR Primers (PRM)
- Consumables
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- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Qubit 1x dsDNA HS Assay Kit (ThermoFisher, Q33230)
- Agilent Technologies DNA 12000 Kit
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Thermal cycler
- Vortex mixer
- Hula mixer (rotator mixer)
- Magnetic rack (e.g. Invitrogen DynaMag-2 Magnet, Cat # 12321D)
- Microfuge
- 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)
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
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Thaw the PCR Primers (PRM) at room temperature, then spin down and place on ice.
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In a 0.2 ml thin-walled PCR tube, prepare the following PCR reaction:
Reagent Stock Final Volume PCR Primer (PRM) 10 μM 0.2 μM 1 μl Nuclease-free water - - 4 μl LongAmp Hot Start Taq 2X Master Mix 2X 1X 25 μl Total - - 30 μl -
Mix by pipetting.
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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.
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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
56°C
65°C15 sec
15 sec
6 min
4Final extension 65°C 10 min 1 Hold 4°C ∞ - -
Resuspend the AMPure XP beads by vortexing.
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Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Add 40 µl of resuspended AMPure XP beads to the 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 in nuclease-free water without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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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.
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Remove the tube from the magnetic rack and resuspend the pellet in 15 µl nuclease-free water.
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Pellet the beads on the magnet until the eluate is clear and colourless.
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Remove and retain 15 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads
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Quantify 1 µl of eluted sample using a Qubit fluorometer - recovery aim >50 ng total.
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Quantify 200 fmol of cDNA from the average fragment size identified using an Agilent Bioanalyzer.
Alternatively, assume an average fragment size of 1 kbp.
Figure: Example amplicon fragment length distribution: 3 biological replicates of 3' cDNA from PBMCs prepared using the 10x genomics 3' gene expression v3.1 kit, processed using the Oxford Nanopore Technologies 3' 10x cDNA protocol. Here the amplicons have been analysed using the Agilent Bioanalyzer 2100 and DNA 12000 kit.