Home Protocols First strand cDNA synthesis OneTaq® RT-PCR Kit

First strand cDNA synthesis OneTaq® RT-PCR Kit

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. Make the RNA/primer/dNTP mix by combining the following components in two sterile RNase-free microfuge tubes.
    Reagent Volume
    Total RNA 1–6 µl*
    Primer 2 µl**
    Nuclease-free Water to a total volume of 8 µl

    * In general, we recommend 1 ng - 1 μg of total RNA or 50 pg - 100 ng of mRNA.
    ** Recommended final primer concentrations: d(T)23VN = 5 μM, Random Primers = 6 μM, specific primers = 0.1-1 μM

  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 containing 8 μl RNA/primer/ dNTP solution and mix well by pipetting up and down.
    Reagent Volume
    M-MuLV Reaction Mix (2X) 10 µl
    M-MuLV Enzyme Mix 2 µl

    Add the following components to the second tube containing the noRT negative control reaction.
    Reagent Volume
    M-MuLV Reaction Mix (2X) 10 µl
    Nuclease-free Water 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 minutes 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 and ready 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.