First Strand cDNA Synthesis (E7530)


 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.


  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.