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  • 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 bufferr   4 µL
      0.1M DTT   2ul
      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)