Second Strand cDNA Synthesis - NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module (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
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    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.