First Strand cDNA Synthesis (E6100)

Protocol

1. Mix the following components in a sterile PCR tube:
   Fragmented mRNA (no less than 50 ng): 13.5 μl
   Random Primers: 1 μl
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   Total volume: 14.5 μl
2. Incubate in a preheated thermal cycler for 5 minutes at 65°C.
3. Spin tube briefly and place on ice
4. To the fragmented mRNA and Random Primers add: 
   5X First Strand Synthesis Reaction Buffer: 4 μl 
   Murine RNase Inhibitor: 0.5 μl 
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   Total volume: 19 μl
5. Incubate in a preheated thermal cycler for 2 minutes at 25°C.
6. Add 1 μl ProtoScript II Reverse Transcriptase to the reaction.
7. Incubate sample as follows:
   10 minutes at 25°C
   50 minutes at 42°C
   15 minutes at 70°C
   Hold at 4°C
8. Place the tube on ice.