2. Spin down cells in 250 ml bottles at 6,000 rpm for 10 minutes at 4 °C.

3. Resuspend cell pellet in 20 ml of P1.

4. Add 20 ml of P2, mix by inverting tubes and pipetting up and down. Incubate at room temperature for 5 minutes.

5. Add 20 ml of chilled P3, mix by inverting 4-6 times or by pipetting up and down. Incubate on ice for 15-20 minutes.

6. Centrifuge at >20,000 g for 30 minutes at 4 °C. Remove supernatant to a fresh tube promptly.

7. Add 0.7 volumes isopropanol, mix well and stand at room temperature for 5 minutes.

8. Centrifuge at >20,000 g for 30 minutes at 4 °C.

9. Discard supernatant. Air dry. Resuspend the DNA in a total volume that is half of the volume of the ultracentrifuge tubes.

10. For each ml of DNA add exactly 1 g of cesium chloride. Mix until dissolved. Add 1% ethidium bromide to a final concentration of approximately 0.75 mg/ml.

11. The final density of the solution should be 1.55 g/ml. Measure the density on a scale. Put the DNA/CsCl mix on the scale, draw 1 ml using a 1 ml pipette. The reading should decrease ~1.55.

12. Transfer the solution to an ultracentrifugation tube using a pasteure pippette, avoid flocculent protein-ethidium bromide complexes.

13. For ultracentrifugation tube with soft wall, fill the tube with TE/CsCl/Ethidium bromide of density 1.55 g/ml.

14. Carefully balance the tubes and seal if necessary.

15. Centrifuge at > 200,000 g in a ultracentrifuge for 16 hours at 25 °C.

16. Stop centrifugation without break to avoid mixing of DNA bands.

17. Two bands should be visible in ordinary light or under a long UV light source. The upper band consists of linear bacterial DNA and nicked circular DNA, the lower one consists of closed circular plasmid DNA.

18. If the tube is sealed, puncture a hole at the top of the tube. Carefully insert a hypodermic needle into the tube just below the lower band and carefully remove the plasmid fraction with a syringe.

19. (optional) Transfer the DNA band to a fresh ultracentrifugation tube, fill with TE/CsCl/Ethidium bromide of density 1.55 g/ml. Centrifuge at > 200, 000 g for 4-16 hours at 25 °C. Remove the plasmid DNA band (there might be only one band visible).

20. Add the DNA/CsCl/Ethidium bromide to a new tube, add equal volume of water-saturated 1-butanol.

21. Mix and let the tube sit at room temperature (or centrifuge at 1,000 g for 3 minutes) until the two phases separated. Remove the top phase (1-butanol).

22. Repeat extraction with water saturated 1-butanol until the DNA solution is colorless.

23. Dilute DNA/CsCl with 3 volumes of TE.

24. Add 1/10 volume of 3 M sodium acetate and 3 volumes of 100% ethanol.

25. Precipitate at -20 °C for >20 minutes.

26. Centrifuge at 10,000 g for 15-20 minutes at 4 °C.

27. Discard supernatant, wash the DNA pellet with 70% ethanol and centrifuge again for 5 minutes at 10,000 g.

28. Air dry and resuspend in 1-2 ml of TE.