First Strand cDNA Synthesis (Standard Protocol) (NEB #M0277)
If denaturation of template RNA is desired, use the following protocol.
- Mix RNA sample and primer d(T)23VN in a sterile RNase-free microfuge tube.
COMPONENT VOLUME 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
- Denature sample RNA/primer for 5 minutes at 65°C. Spin briefly and put promptly on ice.
- Add the following components
- 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.
- 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.
|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|
* 1 ng-1 µg total RNA or 50 pg-100 ng poly(A)-RNA
** AMV Reverse Transcriptase can be used at 42–50°C