First Strand cDNA Synthesis (Quick Protocol) (NEB #M0368)
- 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.
Template RNA up to 1 µg* d(T)23VN (50 µM) or Random Primer Mix (60 µM) 2 µl 5X ProtoScript II buffer 4 µl 0.1 M DTT 2 µl ProtoScript II RT (200 U/µl) 1 µl 10 mM dNTP 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
** ProtoScript II Reverse Transcriptase can be used at 42–48°C.
- Inactivate the enzyme at 65°C for 20 minutes. For downstream PCR
application, the volume of cDNA product should not exceed 1/10 of the PCR