Isolation of Genomic DNA from Bacteria

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