Protocol: Phenol-chloroform Extraction of Prokaryotic DNA
Deoxyribonucleic acid (DNA) extraction is a fundamental, initial procedure in molecular biology and genomics, serving as a critical upstream step for virtually all downstream applications, including PCR, sequencing, and cloning. For simple organisms like bacteria (prokaryotes), the goal is to efficiently separate the total genomic DNA from other cellular components such as proteins, lipids, carbohydrates, and RNA. The extraction process generally follows three basic steps: (1) Lyse the cells to release the contents, (2) Separate the DNA from the unwanted cellular debris, and (3) Isolate and concentrate the purified DNA. Among the oldest and most consistently reliable methods for achieving high yields of high molecular weight DNA is the classic organic extraction method, utilizing a mixture of phenol and chloroform.
Despite the advent of many commercial kits and automated systems, the phenol-chloroform method remains highly relevant, particularly when purity and high yield are paramount, or when working with samples that are challenging for column-based methods. This robust, cost-effective technique is a foundational skill in the microbiology and biochemistry laboratory.
Principle of Phenol-Chloroform Extraction
The phenol-chloroform extraction is a type of liquid-liquid extraction. This method relies on the differential solubility of macromolecules—specifically DNA, proteins, and lipids—in two immiscible liquid phases: an aqueous buffer and an organic solvent mixture. The most common organic mixture used is Phenol:Chloroform:Isoamyl Alcohol (PCI) in a 25:24:1 ratio.
When the PCI mixture is added to an aqueous solution of lysed cells and the phases are thoroughly mixed and separated by centrifugation, the components partition based on their chemical properties. Phenol is a strong protein denaturant. It causes proteins to unfold and separate from the DNA, becoming soluble in the organic phase. Chloroform serves a dual role: it further denatures proteins and, critically, stabilizes the interface between the aqueous and organic layers, making the subsequent removal of the aqueous phase easier and cleaner. Isoamyl alcohol is added to prevent foaming during the mixing step.
Because nucleic acids (DNA and RNA) are highly polar and water-soluble, they remain in the upper aqueous phase. The hydrophobic proteins and lipids are dissolved in the lower organic phase, while a layer of denatured proteins forms a visible white precipitate at the interface. The careful removal of the upper aqueous phase yields a solution highly enriched with nucleic acids and free from most proteins and lipids.
Materials and Pre-Extraction Steps
Successful extraction begins with proper sample preparation and cell lysis. For prokaryotic DNA, a fresh or frozen bacterial cell pellet is typically used, harvested during the late log to early stationary phase for maximum yield. Prior to the organic extraction, the cell walls must be disrupted and the cell contents released. This is achieved using a Lysis Buffer (often containing TE buffer), detergents like Sodium Dodecyl Sulphate (SDS) to disrupt cell membranes, and sometimes a protein-degrading enzyme such as Proteinase K to break down nucleoprotein complexes.
Key reagents for the entire protocol include: TE buffer (Tris-EDTA, pH 8.0) for DNA storage and resuspension; Lysis Buffer (e.g., TE with SDS and Proteinase K); Phenol:Chloroform:Isoamyl Alcohol (PCI, 25:24:1) solution, often pre-buffered to pH 8.0; 7.5 M Ammonium Acetate (NH4OAc) or 3 M Sodium Acetate for salt addition; 100% cold ethanol and 70% ethanol for washing; and a carrier molecule like Glycogen (20 μg/μL) to aid in visualization and precipitation of the DNA pellet.
Phenol-Chloroform Extraction Protocol
The extraction protocol is executed meticulously to ensure maximum purity and minimize shearing of the DNA. The general steps are as follows: First, the bacterial cell pellet is resuspended in Lysis Buffer and incubated, typically at 37°C or 55°C, for 30 minutes to an hour to facilitate complete cell lysis and protein digestion. Next, an equal volume of the Phenol:Chloroform:Isoamyl Alcohol solution is added to the tube containing the lysed sample. The mixture is then vigorously vortexed or shaken by hand for approximately 20 seconds until the two phases are completely mixed and appear cloudy. This intensive mixing maximizes the contact between the organic and aqueous solvents, ensuring proteins are fully denatured and partitioned.
The tube is then centrifuged at high speed (e.g., 14,000–16,000 × g) for 5 minutes at room temperature. This step separates the mixture into three layers: the heavy, lower organic phase (containing proteins and lipids); the light, upper aqueous phase (containing DNA and RNA); and the white, solid interfacial layer (containing denatured proteins). The critical next step is to carefully remove the upper aqueous phase using a micropipette and transfer it to a new, clean tube, being extremely careful not to draw up any of the interfacial material or the lower organic phase, as this would contaminate the DNA sample with phenol and proteins. Often, a second extraction is performed by adding an equal volume of Chloroform:Isoamyl Alcohol (24:1) to the newly isolated aqueous phase, vortexing, and re-centrifuging. Chloroform efficiently removes any residual phenol carried over from the first step, further cleaning the nucleic acid solution before precipitation.
Ethanol Precipitation and Resuspension
To isolate and concentrate the DNA from the aqueous solution, alcohol precipitation is performed. This process requires a high concentration of salt and cold alcohol. First, the salt, such as 7.5 M Ammonium Acetate or 3 M Sodium Acetate, is added to the aqueous phase (typically 1/10th the volume of the sample), followed by the addition of cold 100% ethanol or isopropanol (isopropanol requires less volume, but ethanol is more common). Ethanol is added to 2.5 times the volume of the combined sample and salt solution. The salt neutralizes the negative charge of the DNA backbone, allowing the alcohol to dehydrate the DNA, forcing it out of solution as a visible precipitate. A carrier molecule like glycogen is often added to enhance the visibility of the DNA pellet, especially for low-concentration samples.
The mixture is then incubated in a cold environment, such as at -20°C overnight or on dry ice for at least 30 minutes, to maximize precipitation. Following incubation, the sample is centrifuged at high speed (e.g., 16,000 × g) at 4°C for 20-30 minutes to pellet the DNA. The supernatant (containing the excess salt and alcohol) is carefully discarded, and the white DNA pellet is washed with 70% ethanol. The 70% ethanol wash removes residual salts and small, soluble contaminants. The sample is briefly re-centrifuged, the 70% ethanol is discarded, and the pellet is air-dried briefly to remove all remaining alcohol. Over-drying should be avoided as it makes resuspension difficult. Finally, the dried DNA pellet is resuspended in a small, defined volume of TE buffer or nuclease-free water for storage and quantification. The quality and concentration of the isolated DNA can then be measured using spectrophotometry or agarose gel electrophoresis.
Significance, Advantages, and Safety Considerations
The phenol-chloroform extraction method provides consistently high yields and high purity of DNA suitable for sensitive applications like sequencing. It is highly adaptable and remains the benchmark standard against which commercial kits are often judged. Its primary drawbacks are that it is labor-intensive and utilizes hazardous chemicals. Phenol is highly corrosive and readily absorbed through the skin, while chloroform is a suspected human carcinogen. Consequently, all steps involving these reagents must be strictly performed inside a certified chemical fume hood, and appropriate personal protective equipment (PPE) is mandatory to ensure researcher safety. Despite these safety requirements, the method’s robustness ensures its continued use for challenging samples and high-quality DNA preparations.