First Strand cDNA Synthesis (Standard Protocol) (NEB #M0277)

If denaturation 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.
    Total RNA up to 1 µg*
    d(T)23VN (50 µM) or Random Primer Mix (60 µM) 2 µl
    10 mM dNTP Mix 1 µl
    Nuclease-free H2O To a total volume of 10 µl
  2. Denature sample RNA/primer for 5 minutes at 65°C. Spin briefly and put promptly on ice.
  3. Add the following components
    10X AMV buffer 2 µl
    AMV RT (10 U/µl) 0.2 to 2 µl
    RNase Inhibitor (40 U/µl) 0.2 µl
    Nuclease-free H2O To a total volume of 20 µl

  5. 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.
  6. 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.

* 1 ng-1 µg total RNA or 50 pg-100 ng poly(A)-RNA
** AMV Reverse Transcriptase can be used at 42–50°C