Phenol:Chloroform:Isoamyl Alcohol (PCI): The Classic DNA Extraction Reagent

The Phenol:Chloroform:Isoamyl Alcohol (PCI) mixture is a widely used reagent in molecular biology, especially for the extraction and purification of nucleic acids (DNA and RNA). This powerful organic solvent mixture efficiently separates nucleic acids from proteins and lipids, making it a key component in many genomic and molecular protocols.

What is PCI?

Phenol:Chloroform:Isoamyl Alcohol (PCI) is a liquid-liquid extraction mixture used to isolate DNA or RNA from biological samples. It works by denaturing proteins, separating phases, and allowing nucleic acids to be recovered in a clean aqueous phase.

Typical Composition:

• Phenol: 25 parts (or 50%)

• Chloroform: 24 parts (or 48%)

• Isoamyl Alcohol: 1 part (or 2%)

Ratios may vary slightly depending on the protocol (commonly 25:24:1 by volume).

Role of Each Component

1. Phenol

• Denatures proteins by breaking hydrogen bonds and hydrophobic interactions.

• Helps separate proteins from nucleic acids by making them precipitate or partition into the organic phase.

• Typically equilibrated to pH 7.8–8.0 for DNA extraction and pH 4.5–5.5 for RNA extraction.

2. Chloroform

• Enhances phase separation between the aqueous and organic layers.

• Increases protein denaturation efficiency.

• Helps remove lipids and other contaminants.

3. Isoamyl Alcohol

• Reduces foaming during mixing or centrifugation.

• Aids in clean phase separation and improves clarity of the interface.

Applications in Molecular Biology

1. DNA Extraction

PCI is a standard reagent for isolating high-quality genomic DNA from:

• Bacteria

• Animal and plant tissues

• Blood and cell cultures

It removes proteins, lipids, and other cellular debris, leaving nucleic acids in the aqueous upper phase.

2. RNA Purification

With acidic PCI (lower pH), the method selectively retains RNA in the aqueous phase while DNA and proteins move to the organic phase. It’s widely used in:

• Total RNA extraction

• Removal of genomic DNA contamination

3. Protein Removal

After enzymatic digestion (e.g., proteinase K), PCI is used to:

• Precipitate and denature proteins

• Clean nucleic acid preparations for downstream applications like PCR, sequencing, or cloning

Protocol Overview: DNA Extraction Using PCI

1. Cell Lysis: Cells are lysed using a buffer (e.g., Tris-EDTA with SDS or proteinase K).

2. Add PCI: Mix with equal volume of PCI (25:24:1), then vortex or shake vigorously.

3. Centrifugation: Spin the sample to separate into two phases:

   • Aqueous phase (top): Contains DNA or RNA.

   • Interphase: Precipitated proteins and debris.

   • Organic phase (bottom): Contains denatured proteins, lipids, and phenol.

4. Aqueous Phase Recovery: Carefully transfer the top phase to a new tube.

5. Optional Repeat: Re-extract with phenol or chloroform if further purification is needed.

6. DNA/RNA Precipitation: Precipitate nucleic acids with ethanol or isopropanol, wash with 70% ethanol, and rehydrate in buffer or water.

Safety and Handling

PCI is a hazardous chemical mixture, and appropriate precautions are essential.

Phenol:

• Highly corrosive to skin and mucous membranes.

• Toxic if absorbed through skin or inhaled.

• Use in fume hood with gloves and eye protection.

Chloroform:

• Volatile, toxic, and a potential carcinogen.

• Inhalation can affect the central nervous system and liver.

• Always use in a well-ventilated environment or fume hood.

Isoamyl Alcohol:

• Strong odor; can cause irritation to eyes and respiratory tract.

PPE (Personal Protective Equipment):

• Lab coat

• Nitrile gloves

• Safety goggles

• Use fume hoods for all steps involving PCI

 Storage and Stability

• Store PCI in amber bottles to prevent light-induced degradation.

• Typically stored at 4°C and used within weeks to months.

• Phenol can oxidize over time, turning pink or reddish—this indicates degradation and reduced effectiveness.

• Always check for expiration or discoloration before use.

Alternatives to PCI

Modern molecular biology labs may opt for spin column-based kits (e.g., silica or magnetic bead-based purification) for ease and safety. However, PCI remains widely used due to:

• High purity yield

• Low cost

• Effective removal of proteins and lipids

Final Thoughts

The Phenol:Chloroform:Isoamyl Alcohol mixture is a time-tested reagent in the molecular biology toolbox. It provides efficient, reliable nucleic acid purification, especially when working with tough or dirty samples. While newer techniques may offer convenience and safety, PCI extraction remains the gold standard for many researchers requiring ultra-clean DNA or RNA.

Did You Know?
Despite its harsh nature, PCI extraction is often more effective than commercial kits for samples with high protein content or environmental contaminants—making it the go-to for plant tissues, soil bacteria, and ancient DNA studies.

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