Isolation of genomic DNA from bacteria

Note: This procedure does not work well with Gram + cocci.

1. Transfer 1.5 mL overnight culture to a 1.5 mL microfuge tube, centrifuge for 30 sec, decant supernatant.
    
2. Resuspend cells in 400 mL TE by vortexing, add 50 mL 10 SDS, 50 mL proteinase K (20 mg/mL in TE). Incubate for 1 hour at 37oC.
    
3. Shear DNA by 3-5 passages through a 26 G needle.
    
4. Extract twice with 500 mL phenol:chloroform (1:1), and twice with 500 mL chloroform. 
    
5. Precipitate nucleic acids by adding 25 mL 5 M NaCl and 1 mL 95 EtOH, vortex, centrifuge for 10 min, decant supernatant. Dry pellet.
    
6. Resuspend pellet in 100 mL TE buffer, add 5 mL RNaseA (5 mg/mL in TE), incubate at 37oC for 30 min.
    
7. Precipitate DNA with 40 mL 5M NH4Ac and 250 mL isopropanol, incubate at RT for 5 min.
    
8. Centrifuge for 10 min., wash pellet twice with 70 EtOH, dry pellet, dissolve in 100 mL TE.
    
9. Determine the DNA concentration by measuring the absorbance at 260 nm (1 OD260 = 50 mg/mL) or by comparison of the ethidium bromide staining intensity on a gel to a known amount of uncut lambda DNA.