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  • First Strand cDNA Synthesis (Quick Protocol) (NEB #M0277)

    Thaw components on ice and mix by inverting several times.

    1. Mix the following components and incubate at 42°C for 1 hour. If Random Primer Mix is used, an incubation step at 25°C for 5 minutes is recommended before the 42°C incubation.
       

      COMPONENT

      VOLUME

      Template RNA up to 1 µg*
      d(T)23VN (50 µM) or Random Primer Mix (60 µM) 2 µl
      10X AMV buffer 2 µl
      AMV RT (10 U/µl) 0.2 to 2 µl
      10 mM dNTP Mix 1 µl
      RNase Inhibitor (40 U/µl) 0.2 µl
      Nuclease-free H2O To a total volume of 20 µl

      * 1 ng-1 µg total RNA or 50 pg-100 ng poly(A)-RNA

    2. Inactivate the enzyme at 80°C for 5 minutes. For downstream PCR
      application, the volume of cDNA product should not exceed 1/10 of the PCR
      reaction volume.