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  • Protocol for Use with Previously Fragmented RNA (E7525)

    Starting Material: 50–250 ng of purified mRNA fragmented to 200 nt and cleaned up in 13.5μl of nuclease free water

    First Strand cDNA Synthesis

    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 thermocycler 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 thermocycler 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.
    9. Proceed directly to second strand synthesis using NEBNext mRNA Second Strand Synthesis Module (NEB #E6111).