First Strand cDNA Synthesis (E6116)
Protocol
- Mix the following components in a sterile PCR tube:
Fragmented mRNA: 13.5 μl
Random Primers: 1 μl
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Total volume: 14.5 μl - Incubate in a preheated thermal cycler for 5 minutes at 65°C.
- Spin tube briefly and place on ice.
- 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 - Incubate in a preheated thermal cycler for 2 minutes at 25°C.
- Add 1 μl SuperScript II Reverse Transcriptase to the reaction.
- Incubate sample as follows:
10 minutes at 25°C
50 minutes at 42°C
15 minutes at 70°C
Hold at 4°C - Place the tube on ice.
