The Ultimate Guide to Genomic DNA Isolation
Complete Laboratory Protocols for Bacteria, Plant Tissues, and Animal Cell Lines
Welcome to the comprehensive laboratory guide for genomic DNA extraction. Whether you are working with bacterial cultures, tough plant cell walls, or fragile animal cell lines, this guide covers the principles, precise reagent compositions, and step-by-step procedures you need for pure, high-yield DNA.
Experiment 1: Isolation of Genomic DNA from Bacterial Cells
Aim
To isolate and purify high-molecular-weight genomic DNA from bacterial cells.
Principle
Bacterial genomic DNA isolation involves cell lysis, removal of proteins and contaminants, and DNA precipitation. Bacterial cells are lysed using detergents such as SDS, which disrupt the cell membrane and release cellular components. Proteinase K digests proteins, while RNase A removes RNA contamination. Proteins are removed by phenol–chloroform extraction, and DNA is precipitated using cold alcohol.
1. Overnight Culture
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2. Harvest Pellet
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3. Lysis (SDS + Prot. K)
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4. RNase A Treatment
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5. Phenol-Chloroform
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6. Ethanol Precipitation
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7. 70% Wash & Dry
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8. Dissolve in TE
Materials Required
- Sample: Overnight bacterial culture (e.g., Escherichia coli)
- Reagents: TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), SDS (10%), Proteinase K, RNase A, Phenol:Chloroform:Isoamyl alcohol (25:24:1), Sodium acetate (3 M), Absolute ethanol (cold), 70% ethanol
- Equipment: Microcentrifuge, Water bath (37°C), Micropipettes, Microcentrifuge tubes
Procedure
- Culture Preparation: Inoculate bacterial cells into LB broth medium and incubate overnight at 37°C with shaking.
- Cell Harvesting: Transfer 1.5 mL bacterial culture to a microcentrifuge tube. Centrifuge at 10,000 rpm for 5 minutes. Discard the supernatant and retain the pellet.
- Cell Lysis: Add 500 µL TE buffer to the pellet and resuspend. Add 50 µL SDS (10%) and 10 µL Proteinase K. Incubate at 37°C for 30 minutes.
- RNA Removal: Add 5 µL RNase A. Incubate at 37°C for 15 minutes.
- Phenol–Chloroform Extraction: Add an equal volume of phenol:chloroform:isoamyl alcohol. Mix gently by inversion. Centrifuge at 12,000 rpm for 10 minutes. Transfer the upper aqueous layer containing DNA to a new tube.
- DNA Precipitation: Add 0.1 volume sodium acetate and 2 volumes cold ethanol. Mix gently and incubate at −20°C for 30 minutes.
- Pellet Collection: Centrifuge at 12,000 rpm for 10 minutes. Discard supernatant carefully.
- Washing: Wash DNA pellet with 500 µL of 70% ethanol. Centrifuge for 5 minutes and discard ethanol. Air dry the pellet.
- DNA Dissolution: Dissolve DNA pellet in 50 µL TE buffer. Store at −20°C.
Result: High-molecular-weight genomic DNA is obtained from bacterial cells.
References: Sambrook, J., & Russell, D. W. (2001). Molecular Cloning. | Wilson, K., & Walker, J. (2010). Principles and Techniques.
References: Sambrook, J., & Russell, D. W. (2001). Molecular Cloning. | Wilson, K., & Walker, J. (2010). Principles and Techniques.
Experiment 2: Isolation of DNA from Plant Tissue (CTAB Method)
Aim
To isolate genomic DNA from plant leaf tissue using the CTAB extraction method.
Principle
Plant cells possess cell walls and high levels of polysaccharides and phenolic compounds, which can interfere with DNA extraction. CTAB (Cetyltrimethylammonium bromide) is a cationic detergent that lyses cells and removes polysaccharides. After cell lysis, proteins and other contaminants are removed using chloroform–isoamyl alcohol extraction, and DNA is precipitated with isopropanol or ethanol.
1. Grind in Liquid N₂
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2. Add CTAB Buffer
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3. Incubate at 65°C
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4. Chloroform:IAA
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5. Aqueous Phase
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6. Cold Isopropanol
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7. Wash & Dry
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8. Dissolve in TE
Materials Required
- Sample: Fresh plant leaves (100 mg)
- Reagents: CTAB extraction buffer, Chloroform:Isoamyl alcohol (24:1), Isopropanol (cold), 70% ethanol, TE buffer
- Equipment: Mortar and pestle, Liquid nitrogen, Water bath (65°C), Microcentrifuge
| CTAB Buffer Composition | |
|---|---|
| CTAB | 2% |
| Tris-HCl | 100 mM |
| EDTA | 20 mM |
| NaCl | 1.4 M |
| β-mercaptoethanol | 0.2% |
Procedure
- Tissue Grinding: Take 100 mg fresh leaf tissue. Grind the tissue in liquid nitrogen using mortar and pestle until a fine powder forms.
- Cell Lysis: Transfer powdered tissue to a tube. Add 700 µL CTAB extraction buffer and mix gently.
- Incubation: Incubate at 65°C for 30 minutes. Mix occasionally.
- Organic Extraction: Add equal volume of chloroform:isoamyl alcohol (24:1). Mix gently by inversion. Centrifuge at 12,000 rpm for 10 minutes.
- Transfer Aqueous Phase: Carefully transfer the upper aqueous phase into a new tube.
- DNA Precipitation: Add 0.6 volume cold isopropanol. Mix gently. Incubate at −20°C for 30 minutes.
- Pellet Collection: Centrifuge at 12,000 rpm for 10 minutes. A DNA pellet will appear.
- Washing: Wash pellet with 70% ethanol. Air dry pellet.
- DNA Dissolution: Dissolve DNA pellet in TE buffer.
Result: High-quality plant genomic DNA suitable for PCR and sequencing is obtained.
References: Doyle, J. J., & Doyle, J. L. (1990). Focus. | Wilson, K., & Walker, J. (2010).
References: Doyle, J. J., & Doyle, J. L. (1990). Focus. | Wilson, K., & Walker, J. (2010).
Experiment 3: Isolation of DNA from Animal Cell Lines
Aim
To isolate genomic DNA from cultured animal cells (e.g., HeLa or HEK293).
Principle
Animal cells lack a cell wall, making DNA extraction simpler. Cells are lysed using detergents such as SDS, proteins are digested with Proteinase K, and RNA is removed using RNase A. DNA is purified using phenol–chloroform extraction and precipitated with ethanol.
1. Collect Cells
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2. PBS Wash
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3. Add Lysis Buffer (SDS)
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4. Proteinase K (55°C)
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5. RNase A (37°C)
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6. Phenol:Chloroform
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7. Ethanol Precipitation
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8. Dissolve in TE
Lysis Buffer Composition
- ✔ Tris-HCl: 10 mM
- ✔ EDTA: 10 mM
- ✔ NaCl: 100 mM
- ✔ SDS: 0.5%
Procedure
- Cell Collection: Collect 1–5 × 10⁶ cultured cells in a centrifuge tube. Centrifuge at 3000 rpm for 5 minutes.
- Washing: Wash the pellet with PBS. Centrifuge again and discard supernatant.
- Cell Lysis: Add 500 µL lysis buffer. Add 20 µL SDS. Add 10 µL Proteinase K.
- Incubation: Incubate at 55°C for 1–2 hours until solution clears.
- RNA Removal: Add RNase A. Incubate at 37°C for 20 minutes.
- Phenol–Chloroform Extraction: Add equal volume phenol:chloroform:isoamyl alcohol. Centrifuge at 12,000 rpm for 10 minutes. Transfer aqueous layer to a new tube.
- DNA Precipitation: Add 0.1 volume sodium acetate. Add 2 volumes cold ethanol. Incubate at −20°C for 30 minutes.
- Pellet Collection: Centrifuge at 12,000 rpm for 10 minutes. Wash pellet with 70% ethanol.
- DNA Resuspension: Dissolve pellet in TE buffer.
Result: Purified high-molecular-weight genomic DNA is obtained from animal cell lines.
References: Freshney, R. I. (2016). Culture of Animal Cells. | Green, M. R., & Sambrook, J. (2012).
References: Freshney, R. I. (2016). Culture of Animal Cells. | Green, M. R., & Sambrook, J. (2012).
🧠 Top 10 Viva Voce Questions & Answers
Perfect for practical exams, these questions cover the critical "why" behind the steps in all three extraction methods.
Q1. Why is Liquid Nitrogen used specifically in plant DNA extraction?
A: Plant cells have rigid cellulose cell walls. Liquid nitrogen freezes the tissue, making it brittle and easy to grind into a fine powder without degrading the DNA. It also instantly inactivates endogenous nucleases.
A: Plant cells have rigid cellulose cell walls. Liquid nitrogen freezes the tissue, making it brittle and easy to grind into a fine powder without degrading the DNA. It also instantly inactivates endogenous nucleases.
Q2. What is the role of CTAB in the plant extraction protocol?
A: CTAB is a cationic detergent. It binds to polysaccharides and phenolic compounds (which are abundant in plants) and helps separate them from DNA during the organic extraction phase.
A: CTAB is a cationic detergent. It binds to polysaccharides and phenolic compounds (which are abundant in plants) and helps separate them from DNA during the organic extraction phase.
Q3. Why do we use SDS (Sodium Dodecyl Sulfate) in bacterial and animal cell lysis?
A: SDS is an anionic detergent that dissolves the lipid bilayer of cell membranes and denatures cellular proteins, causing the cell to burst open (lyse) and release its genomic contents.
A: SDS is an anionic detergent that dissolves the lipid bilayer of cell membranes and denatures cellular proteins, causing the cell to burst open (lyse) and release its genomic contents.
Q4. What is the function of Proteinase K in these protocols?
A: Proteinase K is a broad-spectrum serine protease. It rapidly degrades cellular proteins, including histones bound to DNA and highly destructive DNase enzymes, protecting the DNA from degradation.
A: Proteinase K is a broad-spectrum serine protease. It rapidly degrades cellular proteins, including histones bound to DNA and highly destructive DNase enzymes, protecting the DNA from degradation.
Q5. Why is Phenol-Chloroform used, and why in a specific ratio?
A: Phenol denatures proteins, while chloroform ensures phase separation and removes phenol residues. Isoamyl alcohol reduces foaming. The mixture forces denatured proteins into the organic phase, leaving pure nucleic acids in the upper aqueous phase.
A: Phenol denatures proteins, while chloroform ensures phase separation and removes phenol residues. Isoamyl alcohol reduces foaming. The mixture forces denatured proteins into the organic phase, leaving pure nucleic acids in the upper aqueous phase.
Q6. Why is ice-cold absolute ethanol or isopropanol added at the end?
A: DNA is insoluble in alcohol. Adding cold alcohol alters the dielectric constant of the solution, causing the DNA molecules to aggregate and precipitate out of the aqueous solution so they can be pelleted via centrifugation.
A: DNA is insoluble in alcohol. Adding cold alcohol alters the dielectric constant of the solution, causing the DNA molecules to aggregate and precipitate out of the aqueous solution so they can be pelleted via centrifugation.
Q7. What is the purpose of washing the final DNA pellet with 70% ethanol?
A: 70% ethanol washes away residual salts (like sodium acetate or NaCl) that precipitated with the DNA. The remaining 30% water dissolves the salts, while the 70% ethanol keeps the DNA precipitated.
A: 70% ethanol washes away residual salts (like sodium acetate or NaCl) that precipitated with the DNA. The remaining 30% water dissolves the salts, while the 70% ethanol keeps the DNA precipitated.
Q8. Why do animal cells not require the intense physical grinding used for plant cells?
A: Animal cells lack a rigid cell wall; they only have a flexible plasma membrane. Mild chemical detergents (like SDS) are perfectly sufficient to lyse them.
A: Animal cells lack a rigid cell wall; they only have a flexible plasma membrane. Mild chemical detergents (like SDS) are perfectly sufficient to lyse them.
Q9. What is the role of EDTA in the TE buffer and extraction buffers?
A: EDTA is a chelating agent. It binds divalent cations like Mg²⁺, which are essential cofactors for DNase enzymes. By sequestering Mg²⁺, EDTA inactivates DNases, preventing DNA degradation.
A: EDTA is a chelating agent. It binds divalent cations like Mg²⁺, which are essential cofactors for DNase enzymes. By sequestering Mg²⁺, EDTA inactivates DNases, preventing DNA degradation.
Q10. How do you check the purity and quantity of the isolated DNA?
A: Purity and concentration are checked using a UV spectrophotometer (like a NanoDrop). Pure DNA has an A260/A280 absorption ratio of ~1.8. A lower ratio indicates protein contamination, while a higher ratio suggests RNA contamination.
A: Purity and concentration are checked using a UV spectrophotometer (like a NanoDrop). Pure DNA has an A260/A280 absorption ratio of ~1.8. A lower ratio indicates protein contamination, while a higher ratio suggests RNA contamination.
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