• My NEB
  • Print
  • PDF
  • First Strand cDNA Synthesis (E6114)

    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.