Melbourne/Colony PCRl


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  • Applications: Confirm presence of insert in bacterial plasmid

  • Time to complete protocol:
    • Lab time: 5min
    • Waiting time: 4hours
  • Approximate cost of materials: $0.00


Method from primary and secondary reagents

  1. Scrape a colony with a sterile pipette tip and resuspend in 50ul of milliQ water.
  2. Take 3ul of resuspension and make an index plate.
  3. Take 30ul aliquot and boil at 95degrees C for 10 minutes
  4. Use 10ul of resultant supernatant as template in PCR reaction:
    1. 10ul supernatant
    2. 1ul forward primer
    3. 1ul reverse primer
    4. 12 ul GoTaq
  5. Use a PCR machine with the following cycles:
    1. 95C 10min
    2. (92C 1 min 54C 1min 72C 1min) X 30
    3. 72C 10min
    4. 4C hold.
  6. Run the PCR products on a gel.

Primary & secondary Reagents Required including controls
Equipement Required
  • PCR machine
  • Heating block