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 ddH₂O (deionized water)
15. Run 3-4 µL on gel to check quality AND/OR Measure concentration (A260/A280)