First Strand cDNA Synthesis (E6116)

For use with NEBNext® mRNA Library Prep Reagent Set for Illumina® (E6110), NEBNext® mRNA Library Prep Set for SOLiD™ (E6114), NEBNext® mRNA Library Prep Reagent Set for 454™ (E6115), NEBNext® mRNA Library Prep Master Mix Set for 454™ (E6116)

Protocol

  1. Mix the following components in a sterile PCR tube:
    Fragmented mRNA: 13.5 μl
    Random Primers: 1 μl
    -----------------------------------------------------------------------
    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
    -----------------------------------------------------------------------
    Total volume: 19 μl
  5. Incubate in a preheated thermal cycler for 2 minutes at 25°C.
  6. Add 1 μl SuperScript 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.