LARGE SCALE ISOLATION OF PLASMID AND BACTERIAL DNA

1. Grow Escherichia coli in 500 ml of Luria broth (LB) at 37 C with vigorous shaking. To amplify plasmid DNA, add 2.5 ml chloramphenicol (34 mg/ml in ethanol) when culture reaches an OD600 of 0.40.5 and continue vigorous shaking for 1216 hrs.
2. Sediment cells at 5520X g (6000 rpm, JA14 rotor) in two 250 ml centrifuge bottles for 10 min at 4 C.
3. Resuspend cells in 125 ml (0.25 vol) 10 mM Tris (pH 8.0), 10 mM EDTA and resediment (or wash in 1 M NaCl and resediment).
4. Resuspend cells in 10 ml of 15% sucrose, 50 mM Tris (pH 8.0), 50 mM EDTA, 1 mg/ml lysozyme (freshly prepared), and transfer to a 40 ml Oak Ridge tube. Incubate at room temperature for 1060 min. Add RNase to 20 ug/ml. Let stand 10 min.
5. Add 10 ml 0.1% NP40, 50 mM Tris (pH 8.0), 50 mM EDTA. Do not vortex. Mix gently by hand, continuously. If lysis does not occur, incubate at 37 C for 30 min.
6. Centrifuge at 35000X g (17000 rpm, JA20 rotor) for 5060 min at 4 C.
7. Decant supernatant into sterile tube until viscous material above the pellet is reached. Around 18 ml supernatant will be obtained. Add 3.7 g of solid CsCl to four different 10 ml sterile tubes. Add 4 ml supernatant to each tube and mix well by gently inverting tube several times. When mixed, add 0.4 ml ethidium bromide (10 mg/ml) to each tube. Density should be about 1.59 g/ml. Each suspension should be sufficient to fill one 5.1 ml VTi65.2 rotor tube. Load VTi65.2 rotor tube with a 16 gauge needle syringe. Heat seal rotor tubes. Make sure tubes opposite each other in rotor are balanced.
8. Centrifuge at 55000 rpm in VTi65.2 rotor for about 10 hr at 20 C.
9. View bands by illuminating tube by longwavelength uv light. Covalently closed circular DNA is in the lower band. Evenly cut off nipple on top of tube, puncture bottom of tube with 2021 gauge needle and titrate bands out, collecting the DNA band in their respective tubes.
10. Extract out ethidium bromide from DNA sample with equal volume of 5 M NaCl, TE saturated isopropanol. Remove top isopropanol phase and discard. Repeat extraction until solution is clear in color.
11. Add 2 volumes water and 6 volumes 95% ethanol to extracted aqueous phase, then place at 20 C for 3 hours or overnight. Centrifuge at 14600X g (11000 rpm, JA20 rotor) for 30 min at 4 C, decant supernatant, wash with cold 70% ethanol, recentrifuge, decant supernatant, vacuum dry pellet, and resuspend in 100 ul TE.

Note: This procedure does not work too well for isolation of low copy number plasmids in Xanthomonas. It may work if volumes are reduced to fill one rotor tube and plasmid amplification is used.