First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368)
Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community.
- In a sterile microfuge tube add:
RNA solution 1ng-1 µg total RNA or
1-100 ng polyA-selected RNA Primer (50uM dT or 60uM Random Primer Mix)
2 µL 10mM dNTP mix 1 µL nuclease-free H2O to final volume of 12 µL
- (optional) Heat for 3-5 minutes at 65-70°C. Spin briefly and place promptly on ice.
5X ProtoScript II RT Reaction buffer 4 µL 0.1M DTT 2 µL murine RNAse inhibitor (40U/ul) 1 µL ProtoScript II Reverse Transcriptase (200U/ul) 1 µL Final volume 20 µL
- Incubate at 42C for 30min-1hr. If Random Primer Mix is used, an incubation step at 25°C for 5 minutes is recommended before the 42°C incubation.
- Inactivate enzyme at 80°C for 5 minutes.
- Store products at -20°C or proceed to next step(s)