- Materials
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- 16 µl of extracted sample
- Consumables
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- LunaScript™ RT SuperMix Kit (NEB, cat # E3010)
- Applied Biosystems™ Sequenase Version 2.0 DNA Polymerase (Thermo Fisher, cat # 15809896)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Microfuge
- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- 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)
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Prepare the LunaScript™ RT SuperMix Kit and Sequenase Version 2.0 DNA Polymerase according to the manufacturer's recommendations.
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In a clean 0.2 ml thin-walled PCR tube, combine the reagents in the following order:
Reagent Volume Extracted sample 16 µl LunaScript™ RT SuperMix 4 µl Total 20 µl -
Gently pipette mix and spin down the reaction.
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Incubate the sample(s) in the thermal cycler using the following program:
Step Temperature Time Cycles Primer annealing 25°C 2 minutes 1 cDNA synthesis 55°C 10 minutes 1 Heat inactivation 95°C 1 minute 1 Hold 4°C ∞ - -
In a clean 1.5 ml Eppendorf DNA LoBind tube, prepare the first Sequenase master mix by combining the reagents as follows:
Reagent Volume per sample 5X Sequenase reaction buffer 2 µl Nuclease-free water 7.7 µl Sequenase Version 2.0 DNA Polymerase 0.3 µl Total 10 µl -
Mix well by pipette mixing and spin down.
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Add 10 µl of Sequenase master mix to the sample.
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Mix well by pipette mixing and spin down.
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Incubate the reaction at 37°C for 8 minutes.
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In a clean 1.5 ml Eppendorf DNA LoBind tube, prepare the second Sequenase master mix as follows:
Reagent Volume Sequenase Dilution Buffer 0.9 µl Sequenase Version 2.0 DNA Polymerase 0.3 µl Total 1.2 µl -
Mix well by pipette mixing and spin down.
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Add 1.2 µl of the second Sequenase master mix to the sample.
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Mix well by pipette mixing and spin down.
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Incubate the reaction at 37°C for 8 minutes.
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Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Resuspend the AMPure XP beads by vortexing.
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Add 45 µl of resuspended AMPure XP beads to the reaction and mix by gently pipetting.
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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 until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
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Keep the tubes on the magnet and wash the beads in each well with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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Spin down and place the tubes 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 the pellet in 15 µl nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 10 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 each eluted sample using a Qubit fluorometer.