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Star Republic: Guide for Biologists

Reverse transcription

1. Denature RNA secondary structure by heating to 65-70 C for 5 minutes. Place on ice.

2. Prepare a 20 l reaction:

MgCl2, 25mM 4 l
Reverse Transcription 10X Buffer
(1X = 10 mM Tris-HCl pH 8.8, 50 mM KCl, and 0.1% Triton X-100)
2 l
10 mM dNTP mixture 2 l
rRNasin Ribonuclease Inhibitor 0.5 l
AMV Reverse Transcriptase (H.C.) (15 units) 1.5 l
Oligo(dT)15, random, or gene-specific primer (0.5 g) 1 l
RNA (1 g) 2 l
Nuclease-Free ddH2O 7 l
Total volume 20 l

3. Incubate the reaction at 42C for 15 minutes to 1 hour.

4. To analyze the product on an agarose gel, don't heat the sample (or the RNA/cDNA duplex will dissociate).

5. Inactivate the reverse transcriptase by incubating at 95 C for 5 minutes and immediately cool on ice.