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  • Second Strand cDNA Synthesis (E6111)

    Introduction

    Starting Material: Heat inactivated first strand synthesis reaction containing 10-100 ng of first strand cDNA in a 20 μl reaction volume.

    Protocol

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