Synthesize First Strand cDNA

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FSM (First Strand Mix)

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RVT (Reverse Transcriptase)

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1.7 ml microcentrifuge tubes (1 per 96-well sample plate)

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Microseal 'B' adhesive seal

1.

Prepare the following consumables:

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FSM—Invert to mix.

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RVT—Invert to mix before each use.

2.

Save the following IMAP FSS program on the thermal cycler:

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Choose the preheat lid option at 105°C

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Set the reaction volume to 25 µl

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25°C for 5 minutesOpt Option

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50°C for 10 minutesOpt Option

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80°C for 5 minutes

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Hold at 4°C

You can optimize the 25°C and 50°C reverse transcription steps by altering the temperature and incubation times. 50°C is the maximum recommended temperature for this RT enzyme. cDNA synthesis may be improved at lower temperatures (~42°C).

1.

In a 1.7 ml tube, combine the following volumes to prepare FSM. Multiply each volume by the number of samples.

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FSM (9 µl)

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RVT (1 µl)

Reagent overage is included to account for small pipetting errors.

2.

Add 8 µl FSM to each well of the CDNA1 plate.

3.

Seal and shake at 1600 rpm for 1 minute.

4.

Centrifuge at 1000 × g for 1 minute.

5.

Place on the preprogrammed thermal cycler and run the IMAP FSS program.