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Protocol for First-strand cDNA Synth

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  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.

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