First Strand cDNA Synthesis Protocols (E6550)

Introduction

Thaw system components and put on ice. A control reaction without reverse transcriptase is recommended to examine the DNA contamination in the samples.

Protocol

  1. Mix RNA sample and primer d(T)23VN in two sterile RNase-free microfuge tubes.
    Total RNA
    d(T)23VN (50 μM) 2 μl
    nuclease-free H2O variable
    Total Volume 8 μl
  2. Denature RNA for 5 minutes at 70°C. Spin briefly and put promptly on ice. This step is optional. However, it improves the cDNA yield for long messenger RNAs and GC-rich RNA regions.
  3. Add the following components to one tube.
    AMV Reaction Mix 10 μl
    AMV Enzyme Mix 2 μl

    To the negative control tube, add the following:
    AMV Reaction Mix 10 μl
    H2O 2 μl
  4. Incubate the 20 μl cDNA synthesis reaction at 42°C for one hour. If Random Primer Mix is used, an incubation step at 25°C for 5 min is recommended before the 42°C incubation.
  5. Inactivate the enzyme at 80°C for 5 minutes. Dilute reaction to 50 μl with 30 μl H2O for PCR. The cDNA product should be stored at -20°C. For downsteam PCR amplification, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.