Protocol for First-strand cDNA Synthesis
PCR template generation (with M-MuLV Reverse Transcriptase)
- 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
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:
- Incubate at 37°C for 5 minutes. If random primer is used, incubate at 25°C for 5 minutes.
- Add 40 units of M-MuLV Reverse Transcriptase. Incubate the reaction mixture, containing oligo(dT)18 or sequence-specific primer at 37°C for 60 minutes. If using random hexamer primer, incubate at 25°C for 10 minutes and then at 37°C for 60 minutes.
- Stop the reaction by heating at 70°C for 10 minutes. Chill on ice.
- The synthesized cDNA can be amplified by the PCR (see Protocols for PCR using Taq and Pfu DNA Polymerases) without intermediate phenol/chloroform extraction or ethanol precipitation.
Gerard, G.F. and D'Alessio, I.M., Methods in Molecular Biology, 16, Humana Press, Totowa, N.J., 73-93, 1993.
Updated August 23, 2002 13:46