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  • Second Strand cDNA Synthesis

    For use with  NEBNext® mRNA Library Prep Reagent Set for 454™ (E6115) and NEBNext® mRNA Library Prep Master Mix Set for 454™ (E6116)

    Protocol

    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 16 μl sterile water or elution buffer.