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  • First Strand cDNA Synthesis Protocols (E6560)

    Thaw kit components on ice and mix by inverting several times.

    Easy Protocol

    1. Mix the following components and incubate at 42°C for 1 hour. If Random Primer Mix is used, an incubation step at 25°C for 5 minutes is recommended before the 42°C incubation.
      COMPONENT VOLUME
      Template RNA up to 1 µg
      d(T)23 VN 2 µl
      ProtoScript II Reaction Mix (2X) 10 µl
      ProtoScript II Enzyme Mix (10X) 2 µl
      Nuclease-free H2O to a total volume of 20 µl
    2. Inactivate the enzyme at 80°C for 5 minutes. For downstream PCR
      application, the volume of cDNA product should not exceed 1/10 of the PCR
      reaction volume.


    Standard Protocol

    If denature of template RNA is desired, use the following protocol.
    1. Mix RNA sample and primer d(T)23VN in a sterile RNase-free microfuge tube.
      COMPONENT VOLUME
      Total RNA 1–6 µl (up to 1 µg)
      d(T)23 VN (50 µM) 2 µl
      Nuclease-free H2O to a total volume of 8 µl
    2. Denature sample RNA/d(T)23VN for 5 minutes at 65°C. Spin briefly and put promptly on ice.
    3. Add the following components
      ProtoScript II Reaction Mix (2X) 10 μl
      ProtoScript II Enzyme Mix (10X) 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. The cDNA product should be stored at -20°C. In general, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.


    No-RT Negative Control Reaction

    Mix the following components and incubate at 42°C for 1 hour.
    COMPONENT VOLUME
    Total RNA up to 1 µg
    d(T)23 VN (50 µM) 2 µl
    ProtoScript II Reaction Mix (2X) 10 µll
    Nuclease-free H2O to a total volume of 20 µl