Genomic DNA Extraction

Bacterial Genomic DNA Extraction

Protocol

1. Culture 5 ml of the bacteria strain in LB medium until stationary phase (overnight)
2. Centrifuge 2 ml of culture 10 min at room temperature
3. Resuspend the pellet in 540 µl of T10E1 + 30 µl of 10% SDS + 30 µl pronase 20mg/ml
4. Incubate 1h at 37˚C
5. Add 125 µl 4M NaCl and 80 µl of CTAB/NaCl solution
6. Incubate 10 min at 65˚C
7. Extract with same volume of Chloroform/isoamyl alcohol (24:1)
8. Centrifuge 5 min at RT
9. Take the upper phase in a fresh tube and extract with same volume of phenol/( Chloroform/isoamyl alcohol) (1:1)
10. Centrifuge 5 min at RT
11. Add 0.6 volume of isopropanol to the upper phase
12. Leave at -80˚C for 10 min
13. Centrifuge 20 min at 4˚C
14. Wash the pellet with 100 µl of 70% ethanol
15. Dry and resuspend in 100 µl of T10E1 and 1µl RNAse

Notes

• (should obtain 5-7 µg/µl)
• pronase=protease from Streptomyces griseus
• T10E1: TrisHCl pH7.0 10 mM, EDTA 1mM
• CTAB/NaCl solution: 4.1 g NaCl+ 10 g CTAB in 80 ml H2O
  • Stir and heat at 65˚C
  • Adjust to 100 ml (viscous)