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