From 2007.igem.org

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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

Contents 1 Method from primary and secondary reagents 1.1 Primary & secondary Reagents Required including controls 1.2 Equipement Required 1.3 References

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

• milliQ water
• GoTaq
• Forward and Reverse primers
• Bacterial colony

Equipement Required

• PCR machine
• Heating block

References