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Short answer
Phenol:chloroform extraction of DNA is a liquid-phase purification method. A phenol, chloroform and isoamyl alcohol mixture (25:24:1) is added to a lysed sample, and the DNA stays in the upper aqueous layer while proteins and lipids are pulled out. It is inexpensive but labour-intensive, and both main reagents carry occupational exposure limits that set how it must be handled.
Definition and scope
Phenol:chloroform extraction, also called organic extraction, separates nucleic acid from the other material in a lysed sample by mixing the sample with an organic reagent and letting the layers separate. The reagent is a mixture of phenol:chloroform:isoamyl alcohol in a 25:24:1 ratio. According to Thermo Fisher’s protocol reference, phenol is used to denature and extract proteins, while chloroform is used to extract lipids, so the method removes proteins and lipids from the nucleic acid.
This page explains the method and its hazards at the level of a reference. It is not a bench protocol. For the surrounding workflow, including the column-based alternative, see DNA extraction protocol basics. For the RNA-focused variants, see RNA extraction protocol basics. Thermo Fisher marks the method’s protocol page as for research use only and not for use in diagnostic procedures.
How it is used in practice
In Thermo Fisher’s description, the mixture is added to the lysed sample and the tube is spun so the layers separate, with the DNA in the upper aqueous phase. The protocol warns not to carry over any phenol when that layer is removed. The DNA is then recovered from the aqueous phase by ethanol precipitation and an ethanol wash. Centrifuge speed is quoted as relative centrifugal force, which is why how a centrifuge works matters when moving a protocol between rotors.
The same source names the method’s trade-off. It is relatively cost-effective compared with commercial kits or automated systems, which makes it a common choice for researchers with limited resources. Its listed drawbacks are that it is labour-intensive, takes several steps and requires careful handling of hazardous chemicals. Once DNA is recovered, how A260 readings work explains how concentration and purity are checked.
Where it matters for labs: the two reagents side by side
Any lab that adopts the method has to manage two chemicals with different kinds of exposure limit. The values below are taken from the NIOSH Pocket Guide entries for each chemical, checked 2026-10-29.
| Item | Chloroform (CAS 67-66-3) | Phenol |
|---|---|---|
| NIOSH REL | Ca ST 2 ppm (9.78 mg/m3) [60-minute] | TWA 5 ppm (19 mg/m3); C 15.6 ppm (60 mg/m3) [15-minute]; [skin] |
| OSHA PEL | C 50 ppm (240 mg/m3), a ceiling | TWA 5 ppm (19 mg/m3); [skin] |
| IDLH | Ca [500 ppm] | 250 ppm |
| Hazard notes in the NIOSH entry | Listed as a potential occupational carcinogen; liver and kidney cancer in animals | Irritation of eyes, nose and throat; skin burns and dermatitis; target organs include eyes, skin, respiratory system, liver and kidneys |
Two things stand out when the rows are read together. First, chloroform’s recommended limit is a short-term 2 ppm figure carrying a carcinogen flag, set far below its 50 ppm OSHA ceiling, while phenol’s limits are time-weighted averages. Second, phenol carries a skin notation in both the NIOSH and OSHA entries, meaning skin contact is itself a route of exposure and not only inhalation. For why these three kinds of limit differ and which one governs a given lab, see permissible exposure limits versus TLVs and RELs. For containment, see what a fume hood is, and for the waste produced, hazardous waste determination.
Reading the numbers across the columns adds two more points. Chloroform’s NIOSH short-term REL (2 ppm) is 25 times lower than the OSHA ceiling (50 ppm), so a workplace that meets the legal ceiling can still exceed the NIOSH recommendation. And although chloroform’s OSHA ceiling is the larger number, its IDLH value (500 ppm) is twice phenol’s (250 ppm), so the figures cannot be ranked into a single “more dangerous” order from one column alone.
Related methods
Thermo Fisher positions phenol:chloroform extraction against commercial kits and automated systems, which it describes as costlier. Kit-based and column-based purification is covered in the DNA extraction basics guide, and RNA work that uses phenol-based reagents is covered in the RNA guide.
Common misunderstandings
The mixture is not just phenol and chloroform
The 25:24:1 reagent contains three components. Isoamyl alcohol makes up the “1”, so the ratio should be quoted in full when ordering, labelling or recording a protocol.
The two hazards are not interchangeable
The table shows a carcinogen-flagged short-term limit for chloroform and a skin-notated time-weighted limit for phenol. Controls that address one do not automatically address the other.
Cheaper does not mean simpler
Thermo Fisher lists cost-effectiveness as the method’s advantage and in the same passage lists labour intensity, multiple steps and hazardous-chemical handling as its drawbacks.
It is a research method
Thermo Fisher’s protocol page is marked for research use only and not for use in diagnostic procedures.
FAQ
What is phenol:chloroform extraction of DNA?
It is a liquid-phase purification method in which a phenol:chloroform:isoamyl alcohol mixture (25:24:1) is added to a lysed sample so that proteins and lipids are removed from the nucleic acid. Phenol is used to denature and extract proteins, and chloroform is used to extract lipids, according to Thermo Fisher.
What does the 25:24:1 mean?
It is the ratio of phenol to chloroform to isoamyl alcohol in the reagent, so the mixture has three components, not two.
Which layer holds the DNA?
Thermo Fisher’s protocol collects the upper aqueous phase, which holds the DNA, and warns against carrying over any phenol.
Why do labs still use it?
Thermo Fisher describes it as relatively cost-effective compared with commercial kits or automated systems, and a preferred choice for researchers with limited resources. Its drawbacks are that it is labour-intensive, takes several steps and requires careful handling of hazardous chemicals.
What are the exposure limits for the two main reagents?
NIOSH lists chloroform with a short-term REL of 2 ppm (60-minute) and a potential occupational carcinogen flag, and phenol with a 5 ppm time-weighted REL carrying a skin notation. The full figures are in the table above.
Sources
- Thermo Fisher Scientific, Phenol-Chloroform Extraction protocol reference: thermofisher.com, checked 2026-10-29.
- CDC NIOSH Pocket Guide to Chemical Hazards, Chloroform: cdc.gov/niosh/npg/npgd0127.html, checked 2026-10-29.
- CDC NIOSH Pocket Guide to Chemical Hazards, Phenol: cdc.gov/niosh/npg/npgd0493.html, checked 2026-10-29.
