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1. Denature RNA secondary structure by heating to 65-70 °C for 5 minutes. Place on ice.
2. Prepare a 20 µl reaction:
| MgCl2, 25mM | 4 µl |
Reverse Transcription 10X Buffer
(1X = 10 mM Tris-HCl pH 8.8, 50 mM KCl, and 0.1% Triton® X-100) | 2 µl |
| 10 mM dNTP mixture | 2 µl |
| rRNasin® Ribonuclease Inhibitor | 0.5 µl |
| AMV Reverse Transcriptase (H.C.) (15 units) | 1.5 µl |
| Oligo(dT)15, random, or gene-specific primer (0.5 µg) | 1 µl |
| RNA (1 µg) | 2 µl |
| Nuclease-Free ddH2O | 7 µl |
| Total volume | 20 µl |
3. Incubate the reaction at 42°C 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.
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