{"id":584,"date":"2023-10-20T12:17:26","date_gmt":"2023-10-20T12:17:26","guid":{"rendered":"https:\/\/hereditybio.in\/blog\/?p=584"},"modified":"2026-03-24T13:32:53","modified_gmt":"2026-03-24T13:32:53","slug":"bacterial-dna-isolation-by-phenol-chloroform-method","status":"publish","type":"post","link":"https:\/\/hereditybio.in\/blog\/bacterial-dna-isolation-by-phenol-chloroform-method\/","title":{"rendered":"Isolation and Purification of Bacterial Genomic DNA Using the Phenol\u2013Chloroform Extraction Method"},"content":{"rendered":"\n

Heredity Biosciences, Bhubaneswar<\/em><\/strong><\/p>\n\n\n\n

Aim:<\/strong> To isolate high-quality genomic DNA from bacterial cells using the phenol-chloroform method.<\/p>\n\n\n\n

Materials<\/strong><\/p>\n\n\n\n

    \n
  1. Bacterial culture<\/li>\n\n\n\n
  2. TE buffer (10mM Tris, 1mM EDTA, pH 8.0)<\/li>\n\n\n\n
  3. Lysozyme solution (10 mg\/ml)<\/li>\n\n\n\n
  4. Proteinase K solution (20 mg\/ml)<\/li>\n\n\n\n
  5. Sodium dodecyl sulfate (SDS)<\/li>\n\n\n\n
  6. Phenol-chloroform-isoamyl alcohol (25:24:1)<\/li>\n\n\n\n
  7. Isopropanol<\/li>\n\n\n\n
  8. Ethanol (95% or absolute)<\/li>\n\n\n\n
  9. Microcentrifuge tubes<\/li>\n\n\n\n
  10. Sterile distilled water<\/li>\n\n\n\n
  11. RNase (10 mg\/ml)<\/li>\n\n\n\n
  12. 70% ethanol<\/li>\n\n\n\n
  13. DNA extraction kit (optional)<\/li>\n<\/ol>\n\n\n\n

    Procedure<\/strong><\/p>\n\n\n\n

      \n
    1. Start with a fresh bacterial culture (an overnight culture works well). Pellet bacterial cells by centrifuging at 6,000 x g for 5 minutes. Discard the supernatant.<\/li>\n\n\n\n
    2. Resuspend the cell pellet in 500 \u00b5l of TE buffer. Add 25 \u00b5l of lysozyme solution and mix by gentle inversion. Incubate at 37\u00b0C for 30 minutes.<\/li>\n\n\n\n
    3. Add 20 \u00b5l of proteinase K and 200 \u00b5l of 10% SDS. Gently mix by inverting the tube and incubate at 55\u00b0C for 1 hour or until the solution becomes viscous and transparent.<\/li>\n\n\n\n
    4. Add an equal volume of phenol-chloroform-isoamyl alcohol, mix gently, and centrifuge at 12,000 x g for 10 minutes. The DNA is in the aqueous phase.<\/li>\n\n\n\n
    5. Carefully transfer the aqueous phase to a new microcentrifuge tube. Be cautious not to disturb the interphase or organic phase, which may contain proteins and other contaminants.<\/li>\n\n\n\n
    6. Precipitate DNA by adding 0.6 volumes of isopropanol. Gently mix and incubate at -20\u00b0C for at least 30 minutes.<\/li>\n\n\n\n
    7. Centrifuge at 12,000 x g for 15 minutes. The DNA pellet will be visible at the bottom of the tube.<\/li>\n\n\n\n
    8. Carefully pour off the supernatant, ensuring not to disturb the DNA pellet. Wash the pellet with 70% ethanol, centrifuge at 7,500 x g for 5 minutes, and remove the ethanol.<\/li>\n\n\n\n
    9. Air-dry the DNA pellet for 10-15 minutes or until there’s no residual ethanol.<\/li>\n\n\n\n
    10. Resuspend the DNA pellet in 50-100 \u00b5l of sterile distilled water or TE buffer. The DNA is now ready for use or can be stored at -20\u00b0C.<\/li>\n<\/ol>\n\n\n\n

      Notes:<\/strong><\/p>\n\n\n\n