Home Protocols First Strand cDNA Synthesis Protocols (E6560)

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