Endotoxin-Free Plasmid from Spheroplasts

1. Spin down cells from 1 L LB culture for 10 min at 16,000 X g
2. Resuspend pellet in 50 ml of cold 20% sucrose , 50 mM Tris-HCl, pH 8.0 .
3. Add 10 ml of freshly prepared lysozyme (5 mg/ml in 0.25 M Tris, pH 8.0)
4. Add 20 ml of cold 0.25M EDTA, pH 8.
5. Leave on ice for 5 min.
6. Add 20 ml of cold 0.25M Tris-HCl, pH 8.0, incubate at 37 oC for  15 to 30 min.
7. Spin at 4000 g for 10 min, remove viscous supernatant carefully.
8. Resuspend spheroplast pellet in 30 ml of 50 mM glucose/25 mM Tris-HCl, pH 8.0/10 mM EDTA, pH 8.0 containing 100 ug/ml RNase A, or P1 containing purified RNAse or heat-treated RNase.
9. Incubate at 37 oC for 30 min or room temperature o/n.
10. Optional: Add Proteinase K to 100 ug/ml and incubate at 37 oC for 15 min.
11. Add 30 ml of P2. (0.2 M NaOH/1% SDS).
12. Add 30 ml of P3 (5 M KoAc, 11.5% glacial acetic acid) for anion exchange column, or N3 for silica membrane
13. Spin, filter supernatant using coffer paper.
14. Load to plasmid DNA purification column.