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Genomic DNA Isolation from Bacteria (but not Gram + cocci)
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.

This page was created or last modified on 5/13/98 by Jeff Newman .
Assistant Professor Web page: 
Department of Biology Email:
Lycoming College Phone: 717-321-4386
Williamsport PA 17701 Fax: 717-321-4073
© 1998  Jeffrey D. Newman