- Materials
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- Input RNA in 10 mM Tris-HCl, pH 8.0
- Consumables
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- LunaScript™ RT SuperMix Kit (NEB, cat # E3010)
- Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Equipment
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- Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
- Thermal cycler
- Centrifuge capable of taking 96-well plates
- Ice bucket with ice
- Optional equipment
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- PCR-Cooler (Eppendorf)
- PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
- Stepper pipette and tips
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In a clean pre-PCR hood, using a stepper pipette, or a multichannel pipette, add 2 µl of LunaScript™ RT SuperMix to a fresh 96-well plate (RT Plate).
Depending on the number of samples, fill each well per column as follows:
Plate location X24 samples X48 samples X96 samples Columns 1-3 1-6 1-12 -
To each well containing LunaScript reagent of the RT plate, add 8 µl of sample and gently mix by pipetting. If adding less than 8 µl, make up the rest to the volume with nuclease-free water.
Example for X48 samples:
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Seal the RT plate and spin down. Return the plate to ice.
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Preheat the thermal cycler to 25°C.
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Incubate the samples in the thermal cycler using the following program:
Step Temperature Time Cycles Primer annealing 25°C 2 min 1 cDNA synthesis 55°C 10 min 1 Heat inactivation 95°C 1 min 1 Hold 4°C ∞