Team:Calgary/Notebook/Protocols/Process3

From 2011.igem.org

Revision as of 22:05, 27 September 2011 by Emily Hicks (Talk | contribs)


cDNA Synthesis/Reverse Transcription Reaction

  1. Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.
  2. Add the following to a 0.2mL thin-walled PCR tube sitting on ice:
    3. Component Volume
    RNA (40ng – 10ng to 1μg total RNA) variable
    Nuclease free water variable
    qScript cDNA synthesis mix 4 μL
    Final Volume 20 μL
  3. Mix components by gently vortexing and then centrifuge 4s to collect contents.
  4. Incubate in a PCR machine using the following conditions:
    5. 5 min at 25°C
    30 min at 42°C
    5 min at 85°C
    Hold at 4°C
  5. 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).






Retrieved from "http://2011.igem.org/Team:Calgary/Notebook/Protocols/Process3"