REVERSE TRANSCRIPTION (RT) OF DIATOM RNA USING QIAGEN’S QUANTITECT

Note: RNA should always be diluted in RNAse free water.

  1. Please complete the calculations to determine how much RNA template and water need to be added to the RT reaction.
  2. Thaw RNA templates on ice. Make dilutions if necessary. Use RNase free water for dilutions.
  3. Add RNA or RNA+dH2O [total vol.=12μL] to 2μL of gDNA wipeout buffer, already provided in PCR tubes on ice.
  4. Incubate for 2 min at 42ºC in the PCR machine. Then place immediately on ice.
  5. 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.
  6. 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.