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℃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℃for 5 minutes. If random primer is used, incubate at 25℃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℃for 60 minutes. If using random hexamer primer, incubate at 25℃for 10 minutes and then at 37℃for 60 minutes. Stop the reaction by heating at 70℃for 10 minutes. Chill on ice. Note 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. Reference Gerard, G.F. and D'Alessio, I.M., Methods in Molecular Biology, 16, Humana Press, Totowa, N.J., 73-93, 1993. |
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