Team:Calgary/Sandbox/Notebook/Protocols/PlasmidPurificationfromEcoli

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Plasmid Purification from E. coli

Reagents and Materials

  • 3mL overnight cultures
  • P1 : 50 mM TrisHCl (pH 8.0), 10 mM EDTA, 100 µg/ml RNAse A (store at 4°C)
  • P2 : 200 mM NaOH, 1% SDS
  • P3 : 3 M KAc (pH 5.5) (store at 4°C)

Protocol

  1. Grow 3mL O/N culture and use 2 mL for below
  2. Pellet culture into 2 mL microfuge tube
  3. Aspire supernatant and repeat if necessary
  4. Re-suspend pellet in 300 µL P1 (Keep on ice)
  5. *Perform next quickly (<1 min)* Add 300 µL P2 → Invert → Add 300 µL P3
  6. Centrifuge 14000 rpm for 10 min at room temperature
  7. Aliquot the supernatant into 1.5 mL microfuge tube (~600-800 µL)
  8. Add 650 µL isopropanol (RT) → Invert → Incubate for 10 min at room temperature
  9. Centrifuge 14000 rpm for 10 min at 4°C → Aspirate
  10. Wash pellet with 70% cold (-20°C) EtOH (~500 µL)
  11. Centrifuge 14000 rpm for 5 min at 4°C → Aspirate
  12. Air-dry pellet (place tubes upside down for about an hour)
  13. Re-suspend (by flicking) in double distilled water (30 µL)
  14. Leave standing at RT for a few minutes to facilitate dissolving of plasmid in double distilled water
  15. Run 3-4 µL on gel to check quality AND/OR Measure concentration (A260/A280)