PCR on plasmid containing bacterial cells

• grow bacteria in 200-1000 µl LB + anti-biotics over-night
• transfer 3 µl suspension of bacteria into 17 µl bi-destilled water
• boil 10 min
• put on ice 5 min
• centrifuge 5 min
• standard PCR:
  • transfer 3 µl of the supernatant to 47 µl standard PCR mix with flanking primers (e.g. SP6/T7)
  • PCR: (94°C 2 min, 30 x (94°C 30 sec, 50°C 30 sec, 72°C 1 min), 10 min 72°C)
• mini PCR:
  • transfer 1 µl of the supernatant to 9 µl high-efficiency PCR mix with flanking primers (e.g. SP6/T7)
  • PCR: (94°C 2 min, 30 x (94°C 30 sec, 50°C 30 sec, 72°C 30 sec), 10 min 72°C)

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