PROCEDURE :

1. Grow cells in 20 ml of LB containing 10 ul of ampicillin (10 mg/ ml) in the 37 oC shaker for 6-8 hours.

2. Transfer the bacteria to 1L LB solution containing 1 ml Amp in a 2 L flask and incubate overnight at 37 oC.

3. Centrifuge bacteria in a GSA rotor for 10 min at 7,000 rpm. at 4 oC.

4. Discard supernatant. Re-suspend bacteria pellet in 10 ml of Tris- EDTA-glucose solution (soln I) per 1 L solution.

5. Add 1.13 ml of lysozyme solution, incubate in ice for 30 min with occasional gentle mixing.

6. Add 20 ml of NaOH-SDS (soln II), incubate in ice for 10 min with occasional gentle mixing.

7. Add 15 ml of KAc-formic acid solution (soln III), incubate in ice for 30 min with occasional gentle mixing.

8. Spin in GSA rotor for 20 min at 10,000 rpm, 0 oC. (may need to add ~20 ml of soln I per bottle before spin).

9. Using pipette to measure and transfer the supernatant to another bottle. (preferably 150 ml, but a 300 ml one will do since it’s the only kind we have)

10. Add 0.6 volume of isopropanol (Kodak) to the supernatant, mix by hand, immediately spin at 10,000 rpm. for 20 min at 0 oC

11. Re-suspend pellet in 10 ml 1X TE by gentle shaking on a shaker at room temperature.

12. Add 0.3 ml 5 M NaCl solution and mix.

13. Add 2.5 volume of 95% ethanol (-20 oC), mix by hand, and spin at 10,000 rpm for 30 min at 0 oC.

14. Re-suspend pellet in ~10 ml 1X TE by gentle shaking (as in step 11) . Measure volume and transfer to 30 ml Corex tubes.

15. Add CsCl, 1.153 g per ml of sample.

16. Add ethidium bromide, ~0.1 ml EtBr per ml of sample.

17. Spin at 10,000 rpm in a SS-34 rotor for 10 min. Carefully, transfer supernatant to a clean tube, using a disposable syringe. Do not transfer protein precipitates.

18. Load supernatant (using a 3-ml syringe with a G-18 needle to Beckman 5.1-ml quickseal tubes, fill the tube with CsCl/TE solution and seal.

19. Spin at 65,000 rpm for >16 h in VTi 90 rotor at 15 oC.

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20. Collect the lower DNA band (plasmid) under UV light by puncturing below the band using a 3-ml syringe with a G18 needle.

This band should locate at about 2/3 distance from the top of centrifuge tube. The chromosomal DNA band locates about 3 mm above the plasmid DNA band.

21. Combine all plasmid DNA from each centrifuge tube and load it into a clean tube, top it up with CsCl/TE solution and EtBr. Re-centrifuge at the same condition as in step 19 or at 85,000 rpm for 3 h.

22. Collect the lower plasmid DNA band and extract EtBr with 1 volume of NaCl-saturated butanol (top layer) by spinning in JA-17 for 2 min at 2,000 rpm. Extract 4-5 more times until the DNA sample is colorless.

23. Measure volume of the lower phase and transfer to a clean tube.

24. Add 2 volume of water, mix, then 6 volume of absolute ethanol of the total mixture. Precipitate overnight at 4 oC.

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25. Centrifuge 20 min in Sorvall SS-34 rotor at 8,000 rpm, 4 oC.

26. Discard the supernatant and air dry the pellets for ~10 min under the fume hood.

27. Dissolve the pellet in 1 ml 0.5% SDS in TE buffer.

28. Extract with 1 ml phenol/chloroform (top layer) and then 1ml of chloroform. Leave the milky white layer in the middle behind.

29. Add 1/10 volume of 3 M NaAc (pH 5.2) and 2.5 volume ETOH per pellet.

30. Precipitate by placing the mixture in -80 freezer for 12 min, and then centrifuge in the cold room for 20 min.

31. Wash twice with 80% ETOH. Air dry pellets under sterile conditions for at least 1 hr.

32. Dissolve pellets in ~200 ul TE. Store at 4 oC.

Modified by Carol