How to Use Phenol-Chloroform for DNA Purification

Phenol-chloroform extraction effectively removes proteins and lipids from nucleic acid. This results in obtaining relatively pure DNA samples. This method utilizes the differential solubility of DNA, proteins, and lipids in these solvents to isolate DNA from other cellular components. Phenol is used to denature and extract proteins, while chloroform is used to extract lipids. This method can be applied to various sample types, including cells, tissues, and some bodily fluids. It is compatible with both small-scale and large-scale DNA extractions. See below for the phenol-chloroform extraction protocol.

The phenol-chloroform extraction method is relatively cost-effective compared to other commercial kits or automated systems, making it a preferred choice for researchers with limited resources. Setbacks for the phenol-chloroform extraction of DNA include being labor-intensive, requiring several steps, and careful handling of hazardous chemicals. In situations where a high volume of samples need to be processed or time is limited, scientists may opt for automated DNA extraction kits.

• Sample
• TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
• Lysis buffer (e.g., TE buffer with SDS and proteinase K)
• Glycogen (20 μg/μL)
• 7.5 M NH₄OAc (ammonium acetate)
• Ice bucket
• Phenol:chloroform:isoamyl alcohol (ratio of 25:24:1)
• 100% ethanol
• Dry ice or a –80°C freezer
• 70% ethanol

1. Sample preparation: Homogenize the biological sample for complete cell lysis. For cells, resuspend in an appropriate volume of lysis buffer. For tissues, grind in liquid nitrogen and then resuspend in lysis buffer.
2. Cell lysis: Incubate at 55°C for 1-2 hours or until the sample is completely lysed. Add proteinase K to the lysis buffer if necessary.
3. Phenol-chloroform extraction: Add one volume of phenol:chloroform:isoamyl alcohol (25:24:1) to lysed sample, and vortex or shake by hand thoroughly for approximately 20 seconds.
4. Phase separation: Centrifuge at room temperature for 5 minutes at 16,000 × g. Carefully remove the upper aqueous phase and transfer the layer to a fresh tube. Be sure not to carry over any phenol during pipetting.
5. Proceed to "Ethanol precipitation protocol" below.

1. Add the following reagents to the aqueous phase, in the listed order in above table.
2. Place the tube at –20°C overnight to precipitate the DNA from the sample.
   Note: If you wish to continue with the protocol, place the tube in dry ice or at –80°C for at least 1 hour.
3. Centrifuge the sample at 4°C for 30 minutes at 16,000 × g to pellet the cDNA.
4. Carefully remove the supernatant without disturbing the cDNA pellet.
5. Add 150 μL of 70% ethanol. Centrifuge the sample at 4°C for 2 minutes at 16,000 × g. Carefully remove the supernatant.
6. Repeat Step 3 once. Remove as much of the remaining ethanol as possible.
7. Dry the cDNA pellet in a Thermo Scientific SpeedVac concentrator for 2 minutes or at room temperature for 5–10 minutes.
8. Resuspend the cDNA pellet in 50–100 μL of TE buffer or nuclease-free water by pipetting up and down 30–40 times.
9. Measure the DNA concentration and purity using spectrophotometry (e.g., Nanodrop) or by running an aliquot on an agarose gel.