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

Thaw components on ice and mix by inverting several times.

  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)23VN (50 µM) or Random Primer Mix (60 µM) 2 µl
    10X AMV buffer 2 µl
    AMV RT (10 U/µl) 0.2 to 2 µl
    10 mM dNTP Mix 1 µl
    RNase Inhibitor (40 U/µl) 0.2 µl
    Nuclease-free H2O To a total volume of 20 µl

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

  2. Inactivate the enzyme at 85°C for 5 minutes. For downstream PCR
    application, the volume of cDNA product should not exceed 1/10 of the PCR
    reaction volume.