Dandekar & Chandler: Colony PCR for non-E. coli strains

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A pseudomonas colony PCR method that seems to work

  • Scoop up a grain of rice-sized blob of cells (use a stick, blue loops are too big to fit to the bottom of eppi tube)
  • Resuspend in 50 ul sterile dH20
  • Boil for 5 min at 100°C - caps may pop open, so put a heat block thing on top to weigh them down
  • Spin down tubes at 16000g for 5 min
  • Use 5 ul of supernatant in PCR reaction

John's Pa colony PCR method

  • 15 ul H2O in pcr strip tube
  • A dab of the colony
  • Boil 5-10 min (99°C)
  • Add to each tube 13 ul 2x Dreamtaq master mix with primers added in
  • Run program

  • If no amplification but you see smears, you probably have too much DNA:
- Boil cells as above
- Dilute 1:10 into fresh PCR strip tubes and adjust water/master mix appropriately