Organic Extraction Method

Organic extraction is an essential technique in molecular biology, commonly used for isolating nucleic acids, proteins, and cells from complex biological samples. This method relies on the differential solubility of biological molecules in organic solvents and aqueous phases, enabling the effective separation of these biomolecules. While spin columns and magnetic bead-based extraction have grown in popularity, organic extraction remains a valuable approach in many research applications due to its efficiency and cost-effectiveness.

Learn more: DNA protocols
Explore: Bulk reagents

The core principle behind organic extraction is the partitioning of biomolecules into different phases based on their chemical properties. This typically includes an aqueous phase and an organic phase. Organic solvents, such as phenol and chloroform, are added to biological samples, creating a biphasic system. Proteins and lipids tend to partition into the organic phase, while nucleic acids are hydrophilic and remain in the aqueous phase. This process takes advantage of differences in solubility and hydrophobicity, enabling the efficient isolation of desired analytes.

Buffers and pH adjustments are critical in this technique. For example, when extracting DNA, the pH is adjusted to be neutral to slightly basic at around 7–8. This helps ensure the DNA remains in the aqueous phase, while for RNA, acidic conditions are used to keep RNA in the aqueous phase and DNA in the organic layer. After phase separation by centrifugation, the aqueous phase containing nucleic acids can be easily collected, while proteins and other contaminants remain in the organic phase or the interphase.

The choice of purification method depends on the specific properties of the target biomolecule, including its size, charge, solubility, and affinity for certain ligands. Effective biomolecule purification is essential for downstream applications, including structural analysis, functional studies, and therapeutic development. Three main biomolecules studied are nucleic acids, proteins, and cells.

Organic extraction requires a range of reagents to facilitate the phase separation and isolation of biomolecules:

• Phenol—an essential solvent that denatures proteins by disrupting hydrogen bonds, allowing nucleic acids to remain in the aqueous phase
• Chloroform—enhances phase separation by promoting protein and lipid partitioning into the organic phase
• Isoamyl alcohol—often used in conjunction with chloroform to reduce foam formation and stabilize the organic-aqueous interface
• Ethanol/isopropanol—commonly used to precipitate nucleic acids or proteins after extraction, aiding in the recovery and purification of these molecules
• Detergents (e.g., SDS)—solubilize cell membranes, facilitating the release of intracellular contents during cell lysis

View UltraPure Phenol:Chloroform:Isoamyl Alcohol
View Absolute Ethanol, 200 proof, Molecular Biology Grade
View UltraPure Sodium Dodecyl Sulfate (SDS)

Several factors must be optimized for successful organic extraction:

• Sample type—the choice of reagents and method can vary depending on whether the sample is derived from cells, tissues, or bodily fluids. Different samples may require specific protocols to maximize yield and purity.
• pH and buffer conditions—the pH of the extraction buffer determines which molecules are retained in the aqueous phase. For example, acidic phenol is used to extract RNA, while neutral phenol is used for DNA.
• Organic solvent choice—the solvent used impacts the integrity of proteins and the recovery of nucleic acids. Chloroform, for example, can help efficiently separate lipids and proteins from nucleic acids.
• Temperature and centrifugation speed—proper control of temperature and centrifugation speed helps ensure efficient phase separation and minimizes the risk of sample degradation

There are several advantages and disadvantages in choosing organic extraction as a technique to isolate biomolecules from complex mixtures.

Organic extraction is advantageous for yielding high-purity DNA from complex samples, while spin column extraction offers speed, convenience, and safety. Magnetic bead-based extraction stands out for its automation potential, scalability, and high purity, making it suitable for high-throughput and diverse applications. The choice of method depends on the specific requirements of the experiment, including the type of sample, desired purity, and throughput needs. Additionally, organic extraction efficiently separates nucleic acids from proteins, lipids, and other cellular components and is relatively inexpensive, making this method affordable for large sample volumes.

Organic solvents like phenol and chloroform are highly toxic and require careful handling and disposal. The organic extraction process is labor-intensive and involves multiple steps, making it more time-consuming compared to automated methods. If not carefully executed, cross-contamination between phases of organic extraction can reduce the purity of the isolated biomolecules.

• Molecular cloning—high-quality DNA is critical for molecular cloning techniques, where phenol-chloroform extraction remains widely used
• Proteomics—extracting proteins with minimal nucleic acid contamination is essential for proteomic analysis, enzyme assays, and structural studies
• Transcriptomics and RNA-seq—RNA extraction using organic solvents is crucial for isolating high-quality RNA for gene expression profiling and transcriptomic studies
• Cell biology—organic solvents play a key role in cell fractionation, allowing researchers to isolate organelles and membranes for functional studies

• Poor phase separation—inadequate centrifugation or incorrect solvent ratios can prevent proper phase separation. Optimizing centrifugation conditions and solvent ratios can help resolve this issue.
• Low yield—to maximize nucleic acid or protein yield, check if the correct buffer conditions and solvent volumes are being used
• Contamination—if DNA or RNA is contaminated with proteins, additional washing steps can help remove contaminants
• Degraded samples—proper storage, handling, and the use of fresh reagents are crucial to prevent sample degradation

The field of organic extraction is evolving, with new trends focused on improving safety, efficiency, and automation:

• Automation—high-throughput systems are integrating organic extraction protocols, making it easier to process large numbers of samples efficiently
• Greener reagents—there is increasing interest in developing less toxic and more sustainable organic solvents to replace phenol and chloroform
• Improved protocols—novel buffer systems and organic solvent alternatives are being explored to enhance extraction from challenging samples such as low-yield tissues or difficult-to-lyse cells