Isolation of genomic DNA from bacteria

 

<center> <b><u><font><font>Isolation of genomic DNA from bacteria</font> </font> </u> </b></center>

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 m L TE by vortexing, add 50 m L 10% SDS, 50 m L proteinase K (20 mg/mL in TE). Incubate for 1 hour at 37^o C.
   
   
3. Shear DNA by 3-5 passages through a 26 G needle.
   
   
4. Extract twice with 500 m L phenol:chloroform (1:1), and twice with 500 m L chloroform. 
   
   
5. Precipitate nucleic acids by adding 25 m L 5 M NaCl and 1 mL 95% EtOH, vortex, centrifuge for 10 min, decant supernatant. Dry pellet.
   
   
6. Resuspend pellet in 100 m L TE buffer, add 5 m L RNaseA (5 mg/mL in TE), incubate at 37^o C for 30 min.
   
   
7. Precipitate DNA with 40 m L 5M NH₄ Ac and 250 m L isopropanol, incubate at RT for 5 min.
   
   
8. Centrifuge for 10 min., wash pellet twice with 70% EtOH, dry pellet, dissolve in 100 m L TE.
   
   
9. Determine the DNA concentration by measuring the absorbance at 260 nm (1 OD₂₆₀ = 50 m g/mL) or by comparison of the ethidium bromide staining intensity on a gel to a known amount of uncut lambda DNA.