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  • First Strand Synthesis Protocol with Reverse Transcriptase

    Overview

    Materials needed:
    10X RT buffer:
    500 mM Tris-HCl (pH 8.3 @ 25°C)
    750 mM KCl
    30 mM MgCl2
    100 mM DTT

    dNTP mix (2.5 mM each in water titrated by Tris-HCl to pH 7.0)

    Oligo-dT primer (40 µM)

    Random nonamers (40 µM)

    RNase Inhibitor (10 U/µL)

    M-MuLV Reverse Transcriptase (200 units/µL)

    Protocol

    1. In a sterile microfuge tube add:
      RNA solution  0.5-2 µg ( total RNA or 50-100 ng polyA-selected RNA)
      Primer (dT or N9)         2 µL
      dNTP mix                     4 µL
      nuclease-free H2O to final volume of 16 µL
    2. Heat for 3-5 minutes at 65-80°C. Spin briefly and place promptly on ice.
    3. Add:
      10X RT buffer 2 µL
      RNAse inhibitor 1 µL
      M-MuLV Reverse Transcriptase 1 µL
      ______
      final volume 20 µL
    4. Incubate at 42°C for one hour.
    5. Inactivate enzyme at 90°C for 10 minutes.
    6. Store products at -20°C or proceed to next step(s).