Note: RNA should always be diluted in RNAse free water.
- Please complete the calculations to determine how much RNA template and water need to be added to the RT reaction.
- Thaw RNA templates on ice. Make dilutions if necessary. Use RNase free water for dilutions.
- Add RNA or RNA+dH2O [total vol.=12μL] to 2μL of gDNA wipeout buffer, already provided in PCR tubes on ice.
- Incubate for 2 min at 42ºC in the PCR machine. Then place immediately on ice.
- Add 6μL of RT master mix to each of the RNA + gDNA mix. [Final vol. 20μL in each reaction.] Mix by pipetting up and down and store on ice.
- In the PCR machine, incubate for 30 min at 42ºC then incubate for 3 min at 95ºC to inactivate Quantiscript Reverse Transciptase and then hold the temperature at 4ºC.
Store RT reaction on ice and proceed directly with Real-Time PCR, or for long term storage, store RT reactions at -20ºC.