Protocol for Use with Previously Fragmented RNA - NEBNext mRNA First Strand Synthesis Module (E7525)

Protocol B for Use with Previously Fragmented RNA

Starting Material: 100–250 ng of purified mRNA fragmented to 200 nt and cleaned up in 13.5 µl of nuclease free water

  1. First Strand cDNA Synthesis (Non-Directional Reaction Setup)
    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 thermocycler for 5 minutes at 65°C with heated lid set to 105°C. Hold at 4°C.
  3. Spin tube briefly and place on ice 8
  4. To the fragmented mRNA and Random Primers add:
    First Strand Synthesis Reaction Buffer 4 µl
    Murine RNase Inhibitor 0.5 µl
    Total Volume 19 µl
  5. Incubate in a preheated thermocycler for 2 minutes at 25°C.
  6. Add 1 µl ProtoScript II Reverse Transcriptase to the reaction.
  7. Incubate the samples in a preheated thermal cycler (with the heated lid set to ≥ 80°C)
    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.
  9. Proceed directly to second strand synthesis using NEBNext mRNA Second Strand Synthesis Module (NEB #E6111).