Heredity Biosciences, Bhubaneswar

Aim: To isolate high-quality genomic DNA from bacterial cells using the phenol-chloroform method.

Materials

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

Procedure

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.
2. Resuspend the cell pellet in 500 µl of TE buffer. Add 25 µl of lysozyme solution and mix by gentle inversion. Incubate at 37°C for 30 minutes.
3. Add 20 µl of proteinase K and 200 µl of 10% SDS. Gently mix by inverting the tube and incubate at 55°C for 1 hour or until the solution becomes viscous and transparent.
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.
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.
6. Precipitate DNA by adding 0.6 volumes of isopropanol. Gently mix and incubate at -20°C for at least 30 minutes.
7. Centrifuge at 12,000 x g for 15 minutes. The DNA pellet will be visible at the bottom of the tube.
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.
9. Air-dry the DNA pellet for 10-15 minutes or until there’s no residual ethanol.
10. Resuspend the DNA pellet in 50-100 µl of sterile distilled water or TE buffer. The DNA is now ready for use or can be stored at -20°C.

Notes:

• Ensure the complete removal of the organic phase during the phenol-chloroform-isoamyl alcohol extraction steps.
• The optional step 14 can be used for further purification and quantification of the DNA.
• Proper handling of phenol and chloroform is essential, as they are hazardous chemicals.

This method provides high-quality genomic DNA suitable for various molecular biology applications, such as PCR, DNA sequencing, and restriction enzyme digestion.

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