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  • First Strand Synthesis Protocol with AMV Reverse Transcriptase (M0277)

    Protocol

    1. In a sterile microfuge tube add:
      RNA solution - 0.5-2 µg (Total RNA or 50-100 ng polyA mRNA)
      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
      AMV Reverse Transcriptase -1 µL
      H2O - to final reaction volume of 20 µL
    4. Incubate at 42°C for 1 hour.
    5. Inactivate enzyme at 90°C for 5 minutes.
    6. Store products at -20°C or proceed to next step(s) (e.g. PCR).