First Strand cDNA Synthesis (E7420)
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
- 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
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Final volume 20 μl
Note: If you are following recommendations in Appendix A, for longer RNA fragments, incubate for 50 minutes at 42°C.
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
