Phenol Chloroform DNA Extraction: Thermo Fisher Protocol

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to Phenol Chloroform DNA Extraction

Phenol-chloroform extraction is a liquid-liquid phase separation technique used to purify nucleic acids from cellular lysates. The method exploits the differential solubility of DNA, RNA, and proteins in immiscible aqueous and organic phases. When an aqueous cell lysate is mixed with a phenol-chloroform mixture and centrifuged, the solution separates into distinct layers: a lower organic phase containing denatured proteins and lipids, an interphase containing precipitated protein debris, and an upper aqueous phase containing nucleic acids. The aqueous phase is then recovered, and DNA is precipitated with ethanol or isopropanol.

Despite the proliferation of silica membrane spin-column kits, phenol-chloroform extraction remains a gold standard in molecular biology laboratories. The technique offers several advantages that kits cannot match: it yields high-molecular-weight DNA with minimal shearing, it is inexpensive at scale, and it can be adapted to virtually any sample type, from cultured cells to soil microbial communities. For applications requiring very large DNA fragments—such as pulsed-field gel electrophoresis, long-read sequencing on platforms like Oxford Nanopore or PacBio, or genomic library construction—phenol-chloroform extraction is often the method of choice.

What is Phenol Chloroform Extraction?

Phenol-chloroform extraction is a partitioning technique. The principle is straightforward: nucleic acids are hydrophilic and remain in the aqueous phase, while proteins are hydrophobic and partition into the organic phase. Phenol, a weak acid, denatures proteins by disrupting hydrogen bonds and hydrophobic interactions. Chloroform enhances phase separation and stabilizes the interface, while isoamyl alcohol acts as an antifoaming agent and improves the separation of the aqueous and organic layers.

The standard mixture is phenol:chloroform:isoamyl alcohol at a 25:24:1 ratio. This mixture is equilibrated with buffer to a specific pH—typically pH 8.0 for DNA extraction. At this pH, DNA partitions into the aqueous phase, while RNA can be selectively retained in the organic phase or degraded by RNase treatment, depending on the protocol.

Applications in Molecular Biology

Phenol-chloroform extraction is used in a wide range of applications:

• Genomic DNA isolation from bacteria, yeast, plant tissues, and mammalian cells
• Plasmid DNA purification following alkaline lysis
• Removal of proteins from nucleic acid solutions after enzymatic reactions such as proteinase K digestion
• Purification of DNA from agarose gels, though gel reading gel extraction methods often use kits for convenience
• Removal of enzymes after restriction digestion or PCR before downstream applications

The method is particularly valuable when high-molecular-weight DNA is required, as column-based methods inherently shear DNA through membrane binding and elution steps.

Principle of the Technique

The chemistry underlying phenol-chloroform extraction is rooted in the hydrophobic effect and the differential solubility of biomolecules. Understanding the role of each reagent is essential for troubleshooting and for adapting the protocol to unusual sample types.

Role of Phenol

Phenol (C₆H₅OH) is a weak organic acid with a pKa of approximately 10.0. It is a powerful protein denaturant. Phenol disrupts the hydrophobic interactions that maintain protein tertiary structure, causing proteins to unfold. Once denatured, proteins lose their solubility in the aqueous phase and partition into the organic phase. Phenol also disrupts hydrogen bonds between DNA and proteins, releasing nucleic acids into solution.

The pH of phenol is critical. Water-saturated phenol has a pH of approximately 4–5. At acidic pH, DNA partitions into the organic phase while RNA remains in the aqueous phase. This property is exploited in phenol chloroform RNA extraction protocols, where acidic phenol is used to selectively retain DNA in the organic phase while recovering RNA. For DNA extraction, phenol must be equilibrated to pH 7.5–8.0 with Tris-HCl buffer. At this pH, both DNA and RNA remain in the aqueous phase, and proteins partition into the organic phase. The buffering capacity of Tris at pH 8.0 ensures that the aqueous phase maintains a pH at which DNA is fully ionized and hydrophilic.

Role of Chloroform

Chloroform (CHCl₃) serves several functions in the extraction mixture. First, it increases the density of the organic phase, ensuring clean separation from the aqueous phase upon centrifugation. Second, chloroform stabilizes the interface between the aqueous and organic phases, preventing the formation of a diffuse interphase that could trap nucleic acids. Third, chloroform denatures proteins more effectively than phenol alone, particularly proteins such as nucleases that may resist phenol denaturation.

Chloroform also removes phenol from the aqueous phase. After the first extraction with phenol:chloroform:isoamyl alcohol, a second extraction with chloroform alone is often performed to remove residual phenol from the aqueous phase. Phenol carryover can inhibit downstream enzymes such as DNA polymerases and restriction endonucleases, so this step is important for obtaining clean DNA.

Role of Isoamyl Alcohol

Isoamyl alcohol (3-methyl-1-butanol) is included at a 1:24 ratio relative to chloroform. Its primary function is to reduce foaming during mixing. Proteins denatured by phenol tend to accumulate at the interface and can cause emulsification—a stable mixture of aqueous and organic phases that is difficult to separate. Isoamyl alcohol decreases the surface tension at the interface, promoting clean phase separation and preventing emulsion formation. It also aids in the partitioning of proteins into the organic phase.

Thermo Fisher Reagents and Supplies

Thermo Fisher Scientific offers a comprehensive range of reagents and consumables for phenol-chloroform extraction. The following products are commonly used in academic and industrial laboratories.

Phenol:Chloroform:Isoamyl Alcohol (25:24:1)

Thermo Fisher supplies phenol:chloroform:isoamyl alcohol (25:24:1) as a ready-to-use solution, equilibrated with Tris buffer to pH 8.0. The catalog number is 15593031 (100 mL) or 15593049 (400 mL). This product is stabilized with 0.1% 8-hydroxyquinoline, which imparts a yellow color and acts as an antioxidant, preventing phenol oxidation. Oxidized phenol appears pink or brown and can cause DNA degradation; if the solution is discolored, it should be discarded.

The solution is saturated with 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. The EDTA chelates divalent cations such as Mg²⁺ and Ca²⁺, which are cofactors for nucleases, thereby protecting DNA from degradation during extraction.

Phase Lock Gel Tubes

Thermo Fisher offers Phase Lock Gel (PLG) tubes (catalog numbers 733-2478 for 2 mL tubes, 733-2477 for 15 mL tubes) that simplify phase separation. The PLG is a hydrophobic polymer that forms a barrier between the aqueous and organic phases during centrifugation. After centrifugation, the organic phase and interphase are trapped beneath the gel, while the aqueous phase remains above. This eliminates the need to carefully pipette the aqueous phase without disturbing the interphase, reducing the risk of protein contamination. PLG tubes are particularly useful when extracting DNA from samples with high protein content, such as blood or tissue.

Other Required Reagents

Additional reagents for a complete protocol include:

• Proteinase K (catalog number 25530049): A serine protease that digests proteins and nucleases. It is used at a final concentration of 0.1–0.2 mg/mL in lysis buffer.
• RNase A (catalog number EN0531): Used to degrade RNA. RNase A is heat-treated to inactivate DNases. It is added after lysis and incubated at 37°C for 30 minutes.
• Tris-EDTA (TE) buffer (catalog number 12090015): 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. Used to resuspend the final DNA pellet.
• Sodium acetate (catalog number BP333-500): 3 M sodium acetate, pH 5.2, used to provide the salt for ethanol precipitation.
• Ethanol (molecular biology grade, catalog number BP2818-500): 100% and 70% solutions.
• Isopropanol (catalog number BP2618-500): An alternative to ethanol for DNA precipitation.
• Lysis buffer: Typically 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 25 mM EDTA, 0.5% SDS, and 0.1 mg/mL proteinase K. Thermo Fisher offers a ready-to-use Tissue DNA Lysis Buffer (catalog number 4458150).

Step-by-Step Protocol

The following protocol describes genomic DNA extraction from cultured mammalian cells or tissue samples. Volumes are for a starting sample of approximately 10⁶ cells or 25 mg of tissue. Adjust volumes proportionally for other sample sizes.

Sample Preparation and Lysis

1. Harvest cells: For adherent cells, remove the culture medium and wash the monolayer with phosphate-buffered saline (PBS). Add 0.25% trypsin-EDTA and incubate at 37°C for 2–5 minutes until cells detach. Neutralize with serum-containing medium and pellet cells by centrifugation at 300 × g for 5 minutes. For suspension cells, pellet directly. For tissue samples, mince the tissue into small pieces (1–2 mm³) with a sterile scalpel.

1. Resuspend the pellet in 200 µL of PBS. Transfer to a 1.5 mL microcentrifuge tube.

1. Add 400 µL of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 25 mM EDTA, 0.5% SDS, and 0.1 mg/mL proteinase K. Mix by gentle inversion. Do not vortex, as this can shear high-molecular-weight DNA.

1. Incubate at 55–60°C for 2–4 hours (or overnight at 37°C) with occasional gentle mixing. The solution should become clear and viscous as cells lyse and proteins are digested. If the solution remains turbid, add additional proteinase K to a final concentration of 0.2 mg/mL and continue incubation.

1. Add RNase A to a final concentration of 20 µg/mL (e.g., 1 µL of a 10 mg/mL stock) and incubate at 37°C for 30 minutes. This step degrades RNA, which would otherwise coprecipitate with DNA and contaminate the final preparation.

Phase Separation

1. Add an equal volume (600 µL) of phenol:chloroform:isoamyl alcohol (25:24:1, pH 8.0) to the lysate. If using Phase Lock Gel tubes, transfer the lysate to a PLG tube before adding the organic mixture.

1. Mix by inversion for 10–15 seconds until the mixture appears homogeneous and milky. Do not vortex, as this shears DNA and can cause emulsion formation. If the mixture emulsifies, centrifuge briefly and mix more gently.

1. Centrifuge at 12,000 × g for 10 minutes at room temperature. The mixture will separate into three layers: a clear upper aqueous phase containing DNA, a white interphase containing precipitated proteins, and a lower yellow organic phase.

1. Carefully transfer the upper aqueous phase (approximately 500–550 µL) to a fresh tube without disturbing the interphase. If using PLG tubes, simply pipette the aqueous phase from above the gel barrier. Avoid transferring any organic phase or interphase material, as this will contaminate the DNA with phenol and protein.

1. Repeat the extraction by adding an equal volume of phenol:chloroform:isoamyl alcohol to the recovered aqueous phase and repeating steps 7–9. A second extraction is recommended for samples with high protein content.

1. Perform a chloroform-only extraction: Add an equal volume of chloroform to the recovered aqueous phase, mix by inversion, and centrifuge at 12,000 × g for 5 minutes. Transfer the aqueous phase to a fresh tube. This step removes residual phenol, which can inhibit downstream enzymes.

DNA Precipitation and Washing

1. Add 0.1 volumes of 3 M sodium acetate (pH 5.2) to the aqueous phase (e.g., 50 µL for 500 µL of aqueous phase). The sodium acetate provides monovalent cations (Na⁺) that neutralize the negative charge on the DNA phosphate backbone, reducing electrostatic repulsion and allowing DNA to precipitate.

1. Add 2.5 volumes of ice-cold 100% ethanol (e.g., 1.25 mL for 500 µL of aqueous phase). Mix by gentle inversion. A visible DNA precipitate may form immediately, particularly if the DNA concentration is high. For low-concentration samples, precipitate may not be visible.

1. Incubate at -20°C for at least 30 minutes (or overnight for maximum yield). This step allows complete precipitation of DNA. Longer incubation times improve recovery of small DNA fragments.

1. Centrifuge at 12,000 × g for 15 minutes at 4°C. The DNA will form a pellet at the bottom of the tube. The pellet may be invisible or appear as a small white or translucent mass.

1. Carefully remove the supernatant with a pipette, taking care not to dislodge the pellet. Invert the tube on a clean paper towel and allow it to air-dry for 5–10 minutes. Do not over-dry, as this makes the DNA difficult to resuspend.

1. Wash the pellet with 500 µL of 70% ethanol. This removes residual salt and phenol. Centrifuge at 12,000 × g for 5 minutes at 4°C, then remove the supernatant as before.

1. Air-dry the pellet for 5–10 minutes until no visible liquid remains. The pellet will appear translucent.

1. Resuspend the DNA in 50–100 µL of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) or nuclease-free water. Resuspend by gentle pipetting or by incubating at 55°C for 10–15 minutes with occasional tapping. Do not vortex.

1. Quantify the DNA using a Nanodrop Spectrophotometer Thermo Scientific or a fluorometric assay such as Qubit. Assess purity by measuring the A₂₆₀/A₂₈₀ ratio (expected 1.8–2.0 for pure DNA) and the A₂₆₀/A₂₃₀ ratio (expected 2.0–2.2).

Optimizing Yield and Purity

Several factors critically influence the quality and quantity of DNA obtained from phenol-chloroform extraction. Understanding these parameters allows the protocol to be adapted to challenging samples.

pH Considerations

The pH of the phenol solution is the single most important factor determining whether DNA or RNA is recovered. At pH below 7.0, DNA partitions into the organic phase, while RNA remains in the aqueous phase. This property is exploited in the phenol chloroform method of DNA extraction for RNA isolation, where acidic phenol (pH 4.5–5.0) is used. For DNA extraction, phenol must be equilibrated to pH 7.5–8.0. Thermo Fisher's ready-to-use phenol:chloroform:isoamyl alcohol is supplied at pH 8.0, but if you prepare your own phenol, you must equilibrate it with 0.1 M Tris-HCl (pH 8.0) by repeated mixing and phase separation until the aqueous phase reaches pH 8.0.

The pH of the aqueous phase also matters. The lysis buffer should be buffered at pH 8.0 to maintain DNA in its fully ionized, hydrophilic state. At lower pH, DNA becomes protonated and may partition into the organic phase, resulting in poor recovery.

Salt and Alcohol Choices

DNA precipitation requires the presence of monovalent cations to neutralize the phosphate backbone. Sodium acetate (3 M, pH 5.2) is the most common choice, but other salts can be used:

Ethanol and isopropanol are both effective precipitants. Ethanol requires 2.5 volumes and is preferred for small DNA fragments (<500 bp). Isopropanol requires only 0.7 volumes, which is advantageous for large volumes, but it is less volatile and more likely to coprecipitate salt. For high-molecular-weight genomic DNA, ethanol is the better choice.

Handling High-Molecular-Weight DNA

Genomic DNA from eukaryotic cells can exceed 100 kb in length. Such large molecules are extremely viscous and susceptible to shearing. To minimize damage:

• Use wide-bore pipette tips (e.g., 1 mL tips with a 2–3 mm opening) for all transfers.
• Do not vortex at any stage. Mix by gentle inversion or by swirling with a pipette tip.
• Reduce centrifugation speeds to 10,000 × g or lower for phase separation. Higher speeds can shear DNA.
• Avoid repeated pipetting during resuspension. Allow the DNA to dissolve slowly in TE buffer at 4°C overnight, or incubate at 55°C for 10–15 minutes with gentle tapping.
• Consider using a "spooling" method for precipitation: after adding ethanol, gently swirl a glass rod or pipette tip in the solution to wind the DNA around the rod. This recovers high-molecular-weight DNA without centrifugation and avoids the formation of a tight pellet that is difficult to resuspend.

Safety and Waste Disposal

Phenol and chloroform are hazardous chemicals that require careful handling. Both are toxic, and phenol is corrosive and can cause severe chemical burns. Chloroform is a suspected carcinogen and a central nervous system depressant.

Personal Protective Equipment

• Wear a lab coat, nitrile gloves (not latex, which phenol can penetrate), and safety goggles at all times when handling phenol or chloroform.
• Work in a fume hood whenever possible, particularly when opening containers or performing extractions. Phenol has a characteristic sweet odor that indicates significant vapor exposure.
• Change gloves immediately if they become contaminated with phenol or chloroform. Phenol causes a temporary loss of sensation in the skin (a local anesthetic effect), so you may not feel a burn until it is severe.
• Have a spill kit available. For phenol spills, use a polypropylene absorbent material. Do not use paper towels, as phenol can penetrate them.

Chemical Waste Management

• Collect all organic waste (phenol:chloroform:isoamyl alcohol, chloroform, and the organic phases from extractions) in a designated halogenated organic waste container. Do not pour organic solvents down the sink.
• Aqueous waste containing residual phenol should also be collected as hazardous waste, as phenol is toxic to aquatic life.
• Consult the Safety Data Sheet (SDS) for each reagent for specific disposal requirements. Thermo Fisher provides SDS documents for all products on their website.
• Decontaminate work surfaces with 70% ethanol or a commercial decontaminant after completing the extraction. Phenol can leave a residue that is difficult to remove.

Troubleshooting Common Problems

Even experienced researchers encounter problems with phenol-chloroform extraction. The following table summarizes common issues, their causes, and solutions.

Low DNA Yield

Low yield is the most common complaint. The first step is to verify that lysis was complete. If the lysate remains turbid after proteinase K digestion, the protease concentration or incubation time was insufficient. For tough samples such as plant tissues or Gram-positive bacteria, mechanical disruption (bead beating or grinding in liquid nitrogen) may be necessary before chemical lysis.

Loss of DNA during phase separation is another frequent cause. When pipetting the aqueous phase, it is easy to accidentally draw up some of the interphase. Using Phase Lock Gel tubes eliminates this problem. Alternatively, leave a small volume (50 µL) of the aqueous phase behind to avoid the interphase.

Precipitation efficiency depends on salt concentration, alcohol volume, and incubation time. For small DNA fragments (<200 bp), increase the ethanol volume to 3 volumes and incubate at -20°C for at least 1 hour. Adding a carrier such as glycogen (20 µg) or linear polyacrylamide (5 µg) can improve recovery of low-concentration DNA.

Protein or Phenol Carryover

Protein contamination is indicated by an A₂₆₀/A₂₈₀ ratio below 1.8. The most common cause is incomplete proteinase K digestion. Ensure that the enzyme is active (store at -20°C, avoid repeated freeze-thaw cycles) and that the incubation time is adequate. For samples with high protein content, increase the proteinase K concentration to 0.2 mg/mL and extend digestion to overnight.

Phenol carryover is indicated by an A₂₆₀/A₂₃₀ ratio below 2.0. Phenol absorbs at 230 nm, so a low A₂₆₀/A₂₃₀ ratio suggests residual phenol. The chloroform-only extraction step (step 11) is critical for removing phenol. If phenol contamination persists, repeat the chloroform extraction or increase the volume of chloroform used.

RNA Contamination

RNA contamination is visible as a low-molecular-weight smear on an agarose gel. The RNase A digestion step (step 5) should remove most RNA. If RNA persists, ensure that the RNase A is active and that the incubation was performed at the correct temperature (37°C). Some protocols recommend a second RNase A digestion after the first phenol extraction, as the initial digestion may be inhibited by SDS in the lysis buffer.

Comparison with Column-Based Kits

Silica membrane column kits, such as those from Thermo Fisher (GeneJET), QIAGEN (DNeasy), and Zymo Research, have largely replaced phenol-chloroform extraction in routine laboratories. Both methods have distinct advantages and limitations.

Advantages of Phenol Chloroform

• Higher molecular weight DNA: Column-based methods shear DNA as it binds to and elutes from the silica membrane. Phenol-chloroform extraction produces DNA with an average size of 50–100 kb, compared to 20–50 kb for column kits. For applications requiring very large fragments, such as optical mapping or long-read sequencing, phenol-chloroform is superior.
• Lower cost at scale: For large numbers of samples or large sample volumes, the cost of phenol-chloroform reagents is significantly lower than that of column kits.
• Flexibility: The protocol can be scaled up or down, and the reagents can be adapted to unusual sample types. Column kits are optimized for specific sample types and may require additional steps for others.
• No size cutoff: Silica membranes have a lower size limit (approximately 100 bp) below which DNA does not bind efficiently. Phenol-chloroform extraction recovers DNA of all sizes, including small fragments.

Disadvantages and When to Use Kits

• Time and labor: Phenol-chloroform extraction requires multiple centrifugation steps and careful pipetting. Column kits can process 24 samples in under an hour, while phenol-chloroform extraction of the same number of samples may take 2–3 hours.
• Hazardous reagents: Phenol and chloroform require special handling and disposal. Column kits use non-toxic chaotropic salts and are safer for undergraduate laboratories.
• Throughput: Column kits are easily automated on liquid handling platforms. Phenol-chloroform extraction is difficult to automate.
• Consistency: Column kits produce more consistent yields and purity across samples, making them preferable for high-throughput applications such as genotyping or qPCR.

In practice, many laboratories use both methods: column kits for routine PCR and qPCR templates, and phenol-chloroform extraction for applications requiring high-molecular-weight DNA or when working with difficult samples. For RNA isolation, RNA extraction kits are generally preferred over phenol-based methods due to the risk of RNase contamination and the need for specialized reagents such as guanidinium thiocyanate.

Common Pitfalls and Final Checklist

Students frequently make avoidable errors when performing phenol-chloroform extraction. The following pitfalls account for most failed extractions.

Pitfalls to Avoid

1. Using un-buffered phenol: Phenol must be equilibrated to pH 8.0 for DNA extraction. Using acidic phenol will result in DNA partitioning into the organic phase and near-zero recovery.

1. Vortexing the sample: Vortexing shears high-molecular-weight DNA and causes emulsion formation. Always mix by gentle inversion.

1. Pipetting the interphase: The white interphase contains denatured proteins. Transferring any of it to the aqueous phase will contaminate the DNA. Leave a small volume of aqueous phase behind, or use Phase Lock Gel tubes.

1. Skipping the chloroform-only extraction: Residual phenol in the aqueous phase inhibits downstream enzymes. The chloroform-only step is essential for removing phenol.

1. Over-drying the DNA pellet: A completely dry DNA pellet is extremely difficult to resuspend. Air-dry for only 5–10 minutes, and resuspend in TE buffer rather than water (the EDTA in TE protects DNA from nucleases).

1. Using too much salt: Excess sodium acetate can inhibit downstream enzymes. The 70% ethanol wash removes residual salt, so do not skip it.

1. Insufficient proteinase K digestion: For tissue samples, 2 hours at 55°C may not be enough. If the lysate remains turbid, add more enzyme and extend the incubation.

1. Not checking the pH of the aqueous phase: After lysis, the pH should be approximately 8.0. If the lysis buffer was not prepared correctly, the DNA may partition into the organic phase.

Quick Protocol Checklist

• [ ] Phenol:chloroform:isoamyl alcohol (25:24:1) is at pH 8.0
• [ ] Lysis buffer contains proteinase K (0.1–0.2 mg/mL)
• [ ] RNase A added and incubated at 37°C for 30 minutes
• [ ] Mix by inversion, not vortex
• [ ] Centrifuge at 12,000 × g for 10 minutes
• [ ] Transfer aqueous phase without disturbing interphase
• [ ] Repeat phenol:chloroform extraction (2× total)
• [ ] Chloroform-only extraction to remove residual phenol
• [ ] Add 0.1 volumes of 3 M sodium acetate (pH 5.2)
• [ ] Add 2.5 volumes of ice-cold 100% ethanol
• [ ] Incubate at -20°C for at least 30 minutes
• [ ] Centrifuge at 12,000 × g for 15 minutes at 4°C
• [ ] Wash pellet with 70% ethanol
• [ ] Air-dry for 5–10 minutes (do not over-dry)
• [ ] Resuspend in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
• [ ] Quantify by Nanodrop or fluorometric assay

Frequently Asked Questions

What is the purpose of phenol in DNA extraction?

Phenol denatures proteins by disrupting hydrophobic interactions and hydrogen bonds. This causes proteins to unfold and partition into the organic phase, away from the aqueous phase containing DNA. Phenol also dissociates protein-DNA complexes, releasing DNA into solution. The pH of the phenol determines whether DNA (pH 8.0) or RNA (pH 4.5–5.0) is recovered in the aqueous phase.

Why is chloroform used in phenol chloroform extraction?

Chloroform serves three functions: it increases the density of the organic phase for clean separation, it stabilizes the interface between phases, and it enhances protein denaturation. Chloroform also removes residual phenol from the aqueous phase during the final extraction step, preventing phenol carryover that could inhibit downstream enzymes.

What does the 25:24:1 ratio mean in phenol:chloroform:isoamyl alcohol?

The ratio refers to the volumes of the three components: 25 parts water-saturated phenol, 24 parts chloroform, and 1 part isoamyl alcohol. This ratio is optimized for efficient protein denaturation and clean phase separation. The isoamyl alcohol reduces foaming and prevents emulsion formation.

How do I remove RNA during phenol chloroform extraction?

RNA is removed by treating the lysate with RNase A (20 µg/mL final concentration) at 37°C for 30 minutes before the first phenol extraction. RNase A degrades RNA into small fragments that do not precipitate with ethanol. Alternatively, if RNA removal is not critical, it can be left in the sample, but it will coprecipitate with DNA and contaminate the final preparation.

Why is my DNA yield low after phenol chloroform extraction?

Low yield is most commonly caused by incomplete cell lysis, loss of the aqueous phase during extraction, or inefficient precipitation. Ensure that proteinase K digestion is complete (the lysate should be clear), be careful when pipetting the aqueous phase, and consider extending the -20°C incubation during precipitation. For low-concentration samples, add a carrier such as glycogen.

Can I use phenol chloroform extraction for RNA?

Yes, but the protocol must be modified. For RNA extraction, use acidic phenol (pH 4.5–5.0) instead of the pH 8.0 solution used for DNA. At acidic pH, DNA partitions into the organic phase while RNA remains in the aqueous phase. Additional precautions are required to prevent RNase contamination, including the use of RNase-free reagents and diethyl pyrocarbonate (DEPC)-treated water. See the phenol chloroform RNA extraction protocol for details.

What safety precautions should I take when handling phenol?

Wear nitrile gloves, a lab coat, and safety goggles. Work in a fume hood to avoid inhaling phenol vapors. Phenol is corrosive and causes chemical burns; it also has a local anesthetic effect, so burns may not be felt immediately. If phenol contacts skin, wash thoroughly with water and polyethylene glycol (PEG 400) if available. Dispose of phenol waste in a designated halogenated organic waste container.

Key Takeaways

• Phenol-chloroform extraction is a liquid-liquid phase separation technique that purifies DNA by partitioning proteins into an organic phase while DNA remains in the aqueous phase.
• The pH of the phenol is critical: pH 8.0 for DNA extraction, pH 4.5–5.0 for RNA extraction.
• Chloroform stabilizes the phase interface and removes residual phenol; isoamyl alcohol prevents emulsion formation.
• Thermo Fisher supplies ready-to-use phenol:chloroform:isoamyl alcohol (25:24:1, pH 8.0) as catalog number 15593031, along with Phase Lock Gel tubes and other reagents.
• The protocol involves cell lysis with proteinase K, RNase A digestion, repeated phenol:chloroform extractions, a chloroform-only extraction, and ethanol precipitation.
• High-molecular-weight DNA requires gentle handling: mix by inversion, use wide-bore tips, and avoid vortexing.
• Phenol-chloroform extraction yields larger DNA fragments than column-based kits but is more time-consuming and requires handling hazardous chemicals.
• Common problems include low yield (incomplete lysis or precipitation), protein contamination (incomplete digestion or interphase carryover), and phenol carryover (incomplete chloroform extraction).

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