Second Strand cDNA Synthesis

For use with NEBNext® mRNA Library Prep Reagent Set for Illumina® (E6100) and NEBNext® mRNA Library Prep Master Mix Set for Illumina® (E6110).


  1. Add 48 μl Nuclease-Free Water to the First Strand Synthesis reaction.
  2. To the reaction then add:
    10X Second Strand Synthesis Reaction Buffer: 8 μl
    Second Strand Synthesis Enzyme Mix: 4 μl
    Total volume: 80 μl
  3. Mix thoroughly by gentle pipetting.
  4. Incubate in a thermal cycler for 2.5 hours at 16°C.
  5. Purify cDNA using a PCR column purification kit, purifying the sample on one column and eluting in 50 μl sterile water or elution buffer.