Prepare in a sterile tube:
- template RNA: total RNA 0.1-5µg or poly(A)+ mRNA 10ng-0.5µg, or specific RNA 0.01pg-0.5µg
- primer: oligo(dT)18 0.5µg or random hexamer 0.2µg, or sequence-specific 15-20pmol,
- deionized water (nuclease free) up to 11µl.
Incubate the mix at 70°C for 5 minutes and chill on ice. Add the following in the order indicated:
- 5X reaction buffer 4µl,
- 10mM 4 dNTP mix 2µl (1.0mM - final concentration),
- ribonuclease inhibitor 20u,
- deionized water (nuclease free) to 19µl.
Incubate at 37°C for 5 minutes. If random primer is used, incubate at 25°C for 5minutes. Add 40 units of M-MuLV Reverse Transcriptase . Incubate the reaction mixture, containing oligo(dT)18 or sequence-specific primer at37°Cfor 60minutes. If using random hexamer primer, incubate at 25°C for 10 minutes and then at37°Cfor 60 minutes. Stop the reaction by heating at 70°C for 10 minutes. Chill on ice.
Note
- The synthesized cDNA can be amplified by the PCR (seeProtocols for PCR usingTaqandPfuDNA Polymerases) without intermediate phenol/chloroform extraction or ethanol precipitation.
Reference
Gerard, G.F. and D'Alessio, I.M., Methods in Molecular Biology, 16, Humana Press, Totowa, N.J., 73-93, 1993.
