Experimental Protocol for DNA Extraction

DNA extraction is the method by which nuclear DNA is isolated from all other parts of the cell. DNA extraction can be physical or chemical in nature, and can be performed on cultured cells, animal tissues, plants, and bacteria. Though some steps can be adjusted to better suit the desired experimental outcomes, all DNA extraction procedures follow the same general 3-step workflow of lysis, precipitation, and purification.

First, the cells within a sample undergo lysis and DNA is solubilized. Then, membrane lipids, proteins, RNA and all other non-target components of the sample are removed from the sample by chemical or enzymatic means. After, the sample undergoes an elution step to provide a purified DNA product.

The goals of a successful DNA extraction protocol are simple, namely that cellular contaminants and other impurities must be sufficiently removed, and the final resultant DNA be of high purity, quality, and quantity for further use in downstream applications. Below is a simple standard protocol for DNA extraction. Other methods can be used, based on downstream experimental requirements.

Equipment

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1. Microcentrifuge
2. 1.5- or 2.0-mL Microcentrifuge tube
3. 1.5- or 2.0-mL Microcentrifuge spin columns
4. Micropipette
5. Spectrophotometer

Reagents

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Generally, buffers can be purchased premade and many recipes for buffers are readily available online. It is important to optimize buffer conditions geared towards the protocol and desired outcomes. Three standard buffers used in most DNA extraction protocols are:

1. Lysis buffer
   1. The active components of the lysis buffer typically include 0.1 M of a nonionic detergent like sodium dodecyl sulfate (SDS), Tris-Cl, Triton-X and 0.1-0.2 M Ethylene diamine tetraacetic acid (EDTA).
2. Binding buffer
   1. A chaotropic salt-based binding buffer generally supplemented with 0.1-0.2 M EDTA with low alcohol content, designed to optimize binding of gDNA to the spin column.
3. Washing buffer
   1. Typically composed of 10 mM NaCl Tris or TE buffer, supplemented with 1 mM EDTA and 70-95% EtOH

Resources:

Buffer Preparations and Recipes

Protocol

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1. Lysis/homogenization
   1. Tissues (animal or plant)
      1. Grind the tissue into a powder under liquid nitrogen or in an ice bath.
      2. Transfer 20-50 mg tissue to a microcentrifuge tube.
      3. Add 600 uL lysis buffer and incubate for 5-10 min at room temperature.
   2. Cells
      1. The starting cell population should be between 10e5-10e8.
      2. Cells grown in monolayer should be lysed directly in a culture dish. Gently pour off excess media, then add 1 mL lysis buffer.
      3. For cell pellets or suspensions, ensure cells are suspended in at least 200 μL PBS. Add 1 mL lysis buffer to the suspension and gently pipetting the sample. If using <5x10e6 cells, decrease the lysis buffer to 500 μL.
   3. White blood cells in whole blood
      1. The starting white blood cell count should be between 10e5-10e8. Add 1 mL lysis buffer to the cells from more than 500 μL normal whole blood. Gently pipet.
2. To minimize DNA shearing, mix the sample by gentle pipetting or inverting the tube.
   
   Note: Avoid vigorously vortexing the sample.
3. Centrifuge the sample for 10 min at 13,000 g at 4-25 °C.
4. Transfer the supernatant, containing the target DNA to a spin column, and avoid disrupting the pellet.
5. Centrifuge the supernatant 12,000 g for 30 seconds. Discard flow-through.
6. Add 500 μL binding buffer to the spin column. Centrifuge at 12,000 g for 30 s. Discard flow-through.
7. Add 600 μL washing buffer to the spin column. Centrifuge at 12,000 g for 1 min. Discard flow-through.
8. Repeat step 7.
9. Centrifuge the sample for 1 min at 12,000 g and transfer the spin column to a sterile microcentrifuge tube.
10. Add 10-200 μL TE buffer, and incubate at room temperature for 1 min.
11. Centrifuge the sample at 12,000 g for 1 min. The remaining sample within the microcentrifuge tube contains the DNA.
12. The DNA samples can then be stored at -20°C until further use.

Assessing the Quality and Yield of DNA

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Gel electrophoresis can be used to validate the success of a DNA extraction protocol. Spectrophotometry can then be used to measure the concentration of pure DNA produced. The typical absorption peak for nucleic acids is around 260 nm, and an absorbance reading using the A260/A280 ratio (>1.8) is standard for dsDNA. An absorbance ratio of <1.7 indicates poor purity of the resultant DNA, which may mean protein contamination within the sample. For next generation sequencing or other modern applications, fluorescence-based DNA quantitation is generally preferred, since it is more sensitive. DNA quantitation kits or standalone DNA binding dyes are commonly available.

Tool:

DNA Ratio Calculator

Other Types of DNA Extraction

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Determining which DNA extraction method to use is primarily influenced by the intended downstream applications for which the isolated DNA will be used. Besides the quality and quantity of the DNA extracted, other factors to consider are time, cost, yield, laboratory equipment, and the amount of starting material needed for the experiment. Multiple variations are in semi-regular usage:

• Salting out method: RNase A and Proteinase K are used to help precipitate proteins from the sample.
• Organic method: Chloroform and/or phenol are used to denature and precipitate proteins from the sample.
• Adsorption method: A silica-gel membrane is used to adsorb DNA.
• Silica-based method: DNA absorbs to silica beads at a specific pH in presence of specific salts.
• Magnetic separation: DNA binds reversibly to magnetic beads that are pre-coated with DNA-binding antibody.
• Anion exchange technology: Positively charged diethylaminoethyl cellulose (DEAE) functionalized resins capture the negatively charged phosphate backbone of DNA.
• Cesium chloride (CsCl) centrifugation: CsCl is used as a medium to separate DNA in a density gradient.

Further Reading

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Overview of DNA Extraction Methods

Products

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References

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DNA Extraction and Polymerase Chain Reaction
The Evolution of DNA Extraction Methods