Isolation of Genomic DNA from Bacteria

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Isolation of Genomic DNA from Bacteria

  1. Grow 1.5 ml culture of bacteria overnight
  2. Spin 1 minute to collect cells, pour off supernatant
  3. Resuspend pellet in 400 ul TE
  4. Add 50 ul 10% SDS and 50 ul proteinase K (10 mg/ml)
  5. Incubate 1 hour at 37 C with occasional shaking
  6. Siphon up and down through 26 G needle
  7. Add 500 ul saturated phenol:chloroform with isoamyl alcohol (1:24)
  8. Vortex and spin 1 minute
  9. Transfer upper aqueous phase to a new tube
  10. Repeat phenol:chloroform extraction
  11. Extract twice with chloroform/isoamyl alcohol (24:1)
  12. Add TE to final aqueous phase to adjust volume to 500 ul
  13. Add 50 ul 2.5 M NaCl and 1 ml absolute ethanol
  14. Spin 10 minutes, pour off supernatant
  15. Resuspend pellet in 100 ul TE, add 5 ul RNase A (5 mg/ml)
  16. Incubate 30 minute at 37 C
  17. Add 40 ul 5M sodium acetate and 250 ul isopropanol
  18. Place in freezer 10 minutes
  19. Spin 10 minutes
  20. Wash pellet with 500 ul 70% ethanol
  21. Allow pellet to dry
  22. Dissolve pellet in 100 ul of TE or less
  23. Measure absorbance at 260 nm - typical yield is 4 ug to 10 ug