Team:Todai-Tokyo/Protocols/Colony PCR

From 2009.igem.org

UT.png
Home The Team The Project Parts Submitted to the Registry Modeling Notebook Protocols Ethics



the notebook

Colony PCR Protocol

  1. Aliquot into PCR tubes 5µl of MilliQ
  2. Pick single colonies with a toothpick and immerse in the above MilliQ to suspend
  3. Put in PCR machine set at 95ºC for 5 min.
  4. Add to each tube 5µl of the following solution and mix by pipetting
    1. 0.1µl　100µM　5’ primer
    2. 0.1µl　100µM　3’primer
    3. 0.8µl　2.5mM dNTP
    4. 1µl　10x　standard buffer
    5. 2.92µl　MilliQ
    6. 0.08µl　Ex-Taq
  5. PCR using the following program
    1. 95ºC 2 min
    2. 95ºC 30 sec
    3. 52-55ºC 30 sec （Depending on the Tm of the primers）
    4. 72.5ºC 15 sec × （# of kb of DNA to be amplified）
    5. 95ºC 30 sec Repeat 2-4 29 times
    6. 25ºC pause
Retrieved from "http://2009.igem.org/Team:Todai-Tokyo/Protocols/Colony_PCR"