First Strand cDNA Synthesis (E6100)

For use with NEBNext® mRNA Library Prep Reagent Set for Illumina® (E6100)


  1. Mix the following components in a sterile PCR tube:
    Fragmented mRNA (no less than 50 ng): 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 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.