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Note: This procedure does not work well with Gram + cocci.Transfer 1.5 mL overnight culture to a 1.5 mL microfuge tube, centrifuge for 30 sec, decant supernatant.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.Shear
DNA
by 3-5 passages through a 26 G needle.Extract twice with 500 mL phenol:chloroform (1:1), and twice with 500 mL chloroform.
Precipitate nucleic acids by adding 25 mL 5 M NaCl and 1 mL 95 EtOH, vortex, centrifuge for 10 min, decant supernatant. Dry pellet.Resuspend pellet in 100 mL TE buffer, add 5 mL RNaseA (5 mg/mL in TE), incubate at 37oC for 30 min.Precipitate DNA with 40 mL 5M NH4Ac and 250 mL isopropanol, incubate at RT for 5 min.Centrifuge for 10 min., wash pellet twice with 70 EtOH, dry pellet, dissolve in 100 mL TE.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.
