1. Spin down 1.5 ml of overnight culture in 2ml or 1.5ml tube for 1 minute on high.

2. Asprirate supernatant and resuspend cell pellet in 100 µl Solution I (25 mM Tris-HCl, pH 8.0, 10 mM EDTA).

3. Add 200 µl Solution II (0.2 N NaOH, 1.0% SDS) and mix gently by inversion.

4. Add 150 µl Solution III (3M KOAc, pH 4.8 [60 ml 5M KOAc, 11.5 ml HOAc, 28.5 ml H2O]), vortex briefly to mix, and spin for 5 minutes on high.

5. Transfer supernatant to fresh tube containing 500 µl phenol:chloroform, vortex and spin for 5 minutes on high.

6. Transfer aqueous layer to fresh tube containing 1 ml ethanol, mix well by inversion, and spin for 5 minutes on high.

7. Remove supernatant and wash pellet with 100 µl 75% ethanol, spin for 1 minute.

8. Remove as much of ethanol as possible and dry tubes by leaving on bench with lids open for ~5 minutes.

9. Resuspend DNA in 40 µl of 20 µg/ml RNase A H2O.

10. Use for transformation, restriction digestion, sequencing, etc.