How to Use the Phenol Chloroform Extraction Method

Prior to Starting

Know what you’re working with: 
Many reagents in this method are hazardous and it is important to read the MSDS prior to handling these materials.

• Phenol is readily absorbed through the skin and can cause severe burns to the eyes and skin—or your cool new outfit. This effect gets enhanced when phenol is mixed with chloroform.
• Chloroform irritates the skin and eyes, and it is a suspected human carcinogen and reproductive hazard.

Establish special work practices

• Perform procedures involving phenol or chloroform in a chemical fume hood
• Wash hands thoroughly immediately after handling these chemicals 
• Hold the tube and the cap firmly when vortexing to prevent the cap from opening, causing a splash
• Wear the proper personal protective equipment: gloves, eye protection, lab coat, pants, and closed-toed shoes
• Task experienced users with handling these materials
• Store phenol away from strong oxidizers and chloroform away from alkalis
• Discard the reagents and disposable liquid handling equipment in properly labeled hazardous waste bins

Consider some alternatives

There are numerous high-quality commercial kits available for extracting and purifying nucleic acids that serve as safer alternatives to the phenol chloroform extraction method. Check out our wide range of kits in the Sample Preparation Brochure (Read Now).

Reagents Needed

Protocol: Phenol Chloroform Extraction Method

1. Add one volume of phenol:chloroform:isoamyl alcohol (25:24:1) to your sample (Tube 1) (equal parts sample and PCI solution), and vortex for 1 minute.
2. Centrifuge at 16,000 × g at room temperature for 5 minutes. Carefully transfer the upper aqueous phase (containing the DNA) to a fresh 1.5 ml microcentrifuge tube (Tube 2) without transferring any of the lower phenol phase during pipetting.
3. Proceed to ethanol precipitation or continue with the optional protocol below.
   
4. Optional: To maximize the yield of DNA, consider the following steps prior to continuing with ethanol precipitation:
5. Add 200 µL of 10 mM Tris-HCl, pH 8.5  to Tube 1 and vortex for 1 minute.
6. Centrifuge Tube 1 at 16,000 × g at room temperature for 5 minutes 
7. Gently transfer the top DNA-containing aqueous solution from Tube 1 to Tube 2. Be careful not to transfer any of the lower phenol layer to Tube 2. 
8. Add equal volumes of the chloroform/isoamyl alcohol (24:1) solution (Ready-Red™ mixture) to Tube 2 and vortex for 1 minute.
9. Centrifuge Tube 2 at 16,000 × g at room temperature for 5 minutes 
10. Transfer the top aqueous solution  into a fresh tube (Tube 3) without transferring any of the chloroform/isoamyl alcohol phase. 
11. Proceed to ethanol precipitation.

Protocol - Ethanol Precipitation

1. Add the following reagents to the aqueous phase, in the listed order below:
   • Add NH₄OAc to a final concentration of 0.75 M.
   • Add 1 µL of glycogen (20 µg) (Mix)
   • Add 2.5X volume of sample + NH₄OAc of 100% ethanol (Mix)
2. Incubate the tube at –20°C overnight to precipitate the DNA.  Alternatively, you can incubate the tube in dry ice or at –80°C for at least 1 hour.
3. Centrifuge the sample at 4°C for 20-30 minutes at 16,000 × g to pellet the DNA and carefully remove the supernatant without disturbing the pellet.
4. Wash the pellet by adding 150 μL-300 μL of 70% ethanol. Centrifuge the sample at 4°C for 2 minutes at 16,000 × g. Carefully remove the supernatant.
5. Repeat Step 3 and remove as much of the remaining ethanol as possible. Alternatively, you can perform another wash step (step 4) and centrifuge the sample as in step 3. 
6. Air dry the DNA pellet at room temperature for 5–10 minutes.
7. Resuspend the dried DNA pellet in 10 mM Tris-HCl, pH 8.5 by pipetting up and down.