Team:Calgary/Notebook/Protocols/Process3

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cDNA Synthesis/Reverse Transcription Reaction

  • 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:

    • cDNA Synthesis by Reverse Transcription

    Component Volume
    RNA (40ng – 10ng to 1μg total RNA/rxn) variable
    Nuclease free water variable
    qScript cDNA synthesis mix 4 uL
    Final Volume 20 uL
  • Thaw all components (except enzyme), mix thoroughly and centrifuge before use.
  • Mix components by gently vortexing and then centrifuge 4s to collect contents.
  • Incubate in a PCR machine using the following conditions:

    		•
    5 min at 25°C
    30 min at 42°C
    5 min at 85°C
    Hold at 4°C
  • 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).

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