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.

1. Add one volume of phenol:chloroform:isoamyl alcohol (25:24:1) to your sample, and vortex or shakeby hand thoroughly for approximately 20 seconds.


2. 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.


3. Proceed to "Ethanol precipitation" 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 300 μL of TEN buffer by pipetting up and down 30–40 times.


9. Centrifuge briefly to collect the sample, and place the tube on ice.