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  • First Strand cDNA Synthesis (E7420)

    Symbols 

     This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.

     This caution sign signifies a step in the protocol that has two paths leading to the same end point but is dependent on a user variable, like the type of RNA input.

    Prootocol

    1.  Dilute Actinomycin D stock solution (5 μg/μl) to 0.1 μg/μl in nuclease free water for immediate use. 

      Note: Dilute solutions of Actinomycin D are very sensitive to light. In solution, Actinomycin D tends to adsorb to plastic and glass. For these reasons, unused dilute solutions should be discarded and not stored for further use. However, frozen aliquots of a concentrated stock solution (5 μg/μl) are expected to be stable for at least a month at -20°C.

      To the fragmented and primed mRNA (10 μl from section A step 38 or section B step 2) add the following components.

      Murine RNase Inhibitor   0.5 μl 
      Actinomycin D (0.1 μg/μl)   5 μl 
      ProtoScript II Reverse Transcriptase   1 µl
      Nuclease free water   3.5 μl 
      ------------------------------------------------------------
      Final volume   20 μl

      Note: If you are following recommendations in Appendix A, for longer RNA fragments, incubate for 50 minutes at 42°C.
    2.  Incubate the sample in a preheated thermal cycler as follows:
      10 minutes at 25°C
      15 minutes at 42°C
      15 minutes at 70°C
      Hold at 4 °C