Protocol for First-strand cDNA Synth

1. 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.
2. Incubate the mix at 70°C for 5 minutes and chill on ice.
3. 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.
4. Incubate at 37°C for 5 minutes. If random primer is used, incubate at 25°C for 5 minutes.
5. 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.
6. Stop the reaction by heating at 70°C 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.