cDNA Synthesis/Reverse Transcription Reaction
The following protocol is found in the Quanta qScript Reverse Transcription Kit manual, and is used to generate cDNA from isolate RNA (from Pseudomonas spp.) using reverse transcriptase.
- Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.
- Add the following to a 0.2mL thin-walled PCR tube sitting on ice:
- Mix components by gently vortexing and then centrifuge 4s to collect contents.
- Incubate in a PCR machine using the following conditions:
- After completion of cDNA synthesis, us 1/20th of the first-strand reaction (2-4μL) for PCR amplification in water. Also do 2,3, and 4-fold dilutions of each sample to ensure that the PCRs will be within the primer linear range. Do 3 replicates of each PCR reaction. Run PCRs on a real-time PCR machine (e.g. ABI 7900).
|RNA (40ng – 10ng to 1μg total RNA)||variable|
|Nuclease free water||variable|
|qScript cDNA synthesis mix||4 μL|
|Final Volume||20 μL|