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.