Protocol for First-strand cDNA Synthesis

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.