First strand cDNA synthesis OneTaq® RT-PCR Kit
Thaw system components and put on ice. A control reaction without reverse transcriptase is recommended to examine the DNA contamination in the samples.
- Make the RNA/primer/dNTP mix by combining the following components in two sterile RNase-free microfuge tubes.
Reagent Volume Total RNA 1–6 µl (10 pg–2 µg) d(T)23VN (50 µM) 2 µl Nuclease-free Water to a total volume of 8 µl
- Denature RNA for 5 minutes at 70°C. Spin briefly and put promptly on ice. This step is optional. However, it improves the cDNA yield for long messenger RNAs and GC-rich RNA regions.
- Add the following components to one tube containing 8 μl RNA/primer/ dNTP solution and mix well by pipetting up and down.
Reagent Volume M-MuLV Reaction Mix (2X) 10 µl M-MuLV Enzyme Mix 2 µl
Add the following components to the second tube containing the noRT negative control reaction.
Reagent Volume M-MuLV Reaction Mix (2X) 10 µl Nuclease-free Water 2 µl
- 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 4 minutes. Dilute reaction to 50 μl with 30 μl H2O and ready for PCR. The cDNA product should be stored at -20°C. For downsteam PCR amplification, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.