First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368)

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Protocol

  1. In a sterile microfuge tube add:
    RNA solution 1ng-1 µg total RNA or
    1-100 ng polyA-selected RNA Primer (50uM dT or 60uM Random Primer Mix)
    2 µL
    10mM dNTP mix 1 µL
    nuclease-free H2O to final volume of 12 µL


  2. (optional) Heat for 3-5 minutes at 65-70°C. Spin briefly and place promptly on ice.

  3. Add:
    5X ProtoScript II RT Reaction buffer 4 µL
    0.1M DTT 2 µL
    murine RNAse inhibitor (40U/ul) 1 µL
    ProtoScript II Reverse Transcriptase (200U/ul) 1 µL
    Final volume 20 µL


  4. Incubate at 42C for 30min-1hr. If Random Primer Mix is used, an incubation step at 25°C for 5 minutes is recommended before the 42°C incubation.

  5. Inactivate enzyme at 80°C for 5 minutes.

  6. Store products at -20°C or proceed to next step(s)