- Materials
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- PR2 Primer (PR2)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- LongAmp Taq 2X Master Mix (e.g. NEB cat # M0287)
- RiboShredder (Epicentre, cat # RS12500), or RNase Cocktail Enzyme Mix (ThermoFisher, cat # AM2286)
- Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Thermal cycler
- Vortex mixer
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- 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
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Add 1 µl RiboShredder or RNase Cocktail Enzyme Mix (ThermoFisher, cat # AM2286) to the reverse transcription reaction.
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Incubate the reaction for 10 minutes at 37° C in a thermal cycler.
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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 17 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 70% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet. 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 70% 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.
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Repeat the previous step.
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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.
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Remove the tube from the magnetic rack and resuspend pellet in 20 µl nuclease-free water.
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Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Pellet the beads on the magnet until the eluate is clear and colourless.
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Remove and retain 20 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Prepare the following reaction in a 0.2 ml thin-walled PCR tube:
Reagent Volume 2x LongAmp Taq Master Mix 25 μl PR2 Primer, 10 μM 2 μl Reverse-transcribed sample from above 20 μl Nuclease-free water 3 μl Total 50 μl -
Incubate using the following protocol:
Temperature Time No. of cycles 94 °C 1 mins 1 50 °C 1 mins 1 65 °C 15 mins 1 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.
-
Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
-
Spin down the sample and pellet on a magnet. 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.
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.
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Remove the tube from the magnetic rack and resuspend pellet in 21 µl nuclease-free water.
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Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Pellet the beads on the magnet until the eluate is clear and colourless.
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Remove and retain 21 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Analyse 1 µl of the strand-switched DNA for size, quantity and quality.