Synthesize First Strand cDNA

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This step reverse transcribes the hexamer-primed RNA fragments to produce first strand complementary DNA (cDNA).

• FSA (First Strand Synthesis Act D Mix)
• RVT (Reverse Transcriptase)
• 1.7 ml microcentrifuge tube, RNase-free
• Microseal 'B' adhesive film
1. Prepare the following consumables:
• FSA—Invert to mix, and then centrifuge briefly.
• RVT—Invert to mix, and then centrifuge briefly.
2. Save the following FSS program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 25 µl
• 25°C for 10 minutes
• 42°C for 15 minutes
• 70°C for 15 minutes
• Hold at 4°C

Total program time is ~43 minutes.

1. In a 1.7 ml tube on ice, combine the following volumes to prepare First Strand Synthesis (FSS) Master Mix. Multiply each volume by the number of samples.
• FSA (9 µl)
• RVT (1 µl)

Reagent overage is included in the volume.

2. Pipette First Strand Synthesis Master Mix to mix.
3. Remove the seal from the PCR plate.
4. Add 8 µl First Strand Synthesis Master Mix to each well.
5. Pipette 10 times, and then seal.
6. Centrifuge at 280 × g for 10 seconds.
7. Place on the preprogrammed thermal cycler and run the FSS program.

Each well contains a volume of 25 µl.