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  • First Strand cDNA Synthesis (E7530)


    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.

    Protocol

    1. 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
      ProtoScript IIReverse Transcriptase   1 µl
      Nuclease free water   8.5 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
      50 minutes at 42°C
      15 minutes at 70°C
      Hold at 4°C

    3. Immediately, perform second strand synthesis reaction.