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Before starting:

  • Makes sure that screw cap microcentrifuge tubes are available (sterile).
  • Solutions, freshly made and autoclaved:
  1. TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
  2. 3 M Sodium Acetate pH 7.0
  3. 5 M NaCl
  4. 0.5 M EDTA
  5. 20% CTAB
  6. TE saturated Phenol
  7. Chloroform:IAA 24:1
  8. 20 % SDS
  9. 10 mg / ml Lysozyme
  10. 20 mg / ml Proteinase K
  11. liquid nitrogen for freeze thaw

Extraction protocoll

  1. pellet cells
  2. add 467 ul TE and re-suspend pellet
  3. freeze thaw with liquid nitrogen 5 times
  4. add 153 ul 0.5M EDTA
  5. add 38 ul 10% SDS
  6. add 10 ul of lysozyme solution
  7. incubate for 30 minutes
  8. add 10 ul of proteinase K solution
  9. incubate for 30 minutes
  10. add 120 ul of 5M NaCl
  11. add 100 ul of 10% CTAB
  12. incubate at 65 deg C for one hour
  13. extract with phenol/chloroform/isoamyl acohol (25:24:1)
  14. precipitate DNA with 0.6 volumes of isopropanol
  15. wash pellet with 70% ethanol
  16. dry pellet
  17. take pellet up in 50 ul of TE buffer (more buffer may be needed if DNA yield is high)
 
dna_extraction/dna_extraction_from_anaerobic_cultures_-_amy_callaghan.txt · Last modified: 2008/03/20 10:54 by 129.15.161.177