Genomic DNA Extraction

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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)
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