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What are the steps of DNA extraction?
Posted November 9, 2020
Answer
There are 3 basic steps involved in DNA extraction, that is, lysis, precipitation and purification. In lysis, the nucleus and the cell are broken open, thus releasing DNA. This process involves mechanical disruption and uses enzymes and detergents like Proteinase K to dissolve the cellular proteins and free DNA.
The other step, which is known as precipitation, separates the freed DNA from the cellular debris. It involves use of sodium (Na+) ions to neutralize any negative charge in DNA molecules, making them less water soluble and more stable. Alcohol (e.g isopropanol or ethanol) is then added, causes precipitation of DNA from the aqueous solution since it does not dissolve in alcohol.
After separation of DNA from aqueous solution, it is then rinsed with alcohol, a process known as purification. Purification removes all the remaining cellular debris and unwanted material. Once the DNA is completely purified, it is usually dissolved in water again for convenient storage and handling.
The other step, which is known as precipitation, separates the freed DNA from the cellular debris. It involves use of sodium (Na+) ions to neutralize any negative charge in DNA molecules, making them less water soluble and more stable. Alcohol (e.g isopropanol or ethanol) is then added, causes precipitation of DNA from the aqueous solution since it does not dissolve in alcohol.
After separation of DNA from aqueous solution, it is then rinsed with alcohol, a process known as purification. Purification removes all the remaining cellular debris and unwanted material. Once the DNA is completely purified, it is usually dissolved in water again for convenient storage and handling.
Protocol: Genomic DNA Extraction
The following is a sample protocol for the extraction of genomic DNA from cell culture. Sample Size: Start with 1 x 105 to 5 x 106 cells.
- Harvest cells from the culture vessel.
- Pellet cells by centrifugation at 1000 x g for 1 minute and discard supernatant.
- Resuspend the cell pellet in 100 µL cold PBS and mix by pipetting up and down repeatedly.
- Add 100 µL of cell lysis buffer (see recipe) and 2 µL of 20 mg/mL proteinase K and mix by vortexing.
- (Optional): Add 3 µL of RNase A
- Incubate in a thermal mixer at 56°C for 1 hour with agitation at 1400 rpm. Alternatively if a thermal mixer is not available, use a 56°C water bath or heating block and vortex occasionally.
- Thoroughly extract the samples with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and mix well by inverting the tube until the phases are completely mixed.
- Spin at max speed for 5 minutes, and carefully transfer the upper aqueous layer to a fresh Eppendorf tube.
- To precipitate DNA add 1 mL of 100% EtOH (room temperature), close tube and gently invert until DNA precipitate forms.
- Incubate the tube at room temperature for 15 – 30 minutes.
- Spin at max speed for 5 minutes and carefully remove and discard supernatant.
- Wash the DNA pellet with 1 mL 70% EtOH (-20°C) and invert several times.
- Spin at max speed for 2 minutes, and carefully remove and discard supernatant.
- Dry the DNA pellet at room temperature overnight or dry using a vacuum concentrator
- Resuspend DNA pellet in an appropriate volume of TE buffer.
DNA Quantitation
DNA concentration can be determined either by absorbance or fluorescence. To determine DNA concentrations using the UV-absorbance method measure the absorbance of the DNA sample at 260 nm with a spectrophotometer.
Fluorescence methods determine DNA concentration by using double-stranded DNA binding dyes, such as Helixyte™ Green (AAT Bioquest Cat No. 17597) that fluoresce when bound to dsDNA, or DNA quantitation kits, such as the Portelite™ Fluorimetric High Sensitivity DNA Quantitation Kit (Cat No. 17661) or the Portelite™ Fluorimetric DNA Quantitation Kit with Broad Dynamic Range (Cat No. 17665). The fluorescence intensity is measured using a fluorometer, such as the CytoCite™ BG100 portable fluorometer from AAT Bioquest (Cat No. CBG100). Of the two methods, fluorescence-based DNA quantitation is more sensitive and generally used to quantify DNA for next generation sequencing.
Helixyte™ Green
Fluorescence methods determine DNA concentration by using double-stranded DNA binding dyes, such as Helixyte™ Green (AAT Bioquest Cat No. 17597) that fluoresce when bound to dsDNA, or DNA quantitation kits, such as the Portelite™ Fluorimetric High Sensitivity DNA Quantitation Kit (Cat No. 17661) or the Portelite™ Fluorimetric DNA Quantitation Kit with Broad Dynamic Range (Cat No. 17665). The fluorescence intensity is measured using a fluorometer, such as the CytoCite™ BG100 portable fluorometer from AAT Bioquest (Cat No. CBG100). Of the two methods, fluorescence-based DNA quantitation is more sensitive and generally used to quantify DNA for next generation sequencing.
Helixyte™ Green
Assesing DNA Purity
Purity of DNA and RNA samples can be assessed by taking the ratio of absorbance at 260 nm and 280 nm (A260/A280). A ratio of ∼1.8 indicates a pure DNA sample, while a ratio of ∼2.0 indicates a pure RNA sample. If the ratio is higher or lower, it may indicate the presence of contaminates (e.g. protein, phenol, etc.) which also absorb at or near 280 nm. Strong absorbance around 230 nm can indicate that organic compounds or chaotropic salts are present in the purified DNA. A ratio of 260 nm to 230 nm can help evaluate the level of salt carryover in the purified DNA. The lower the ratio, the greater the amount of thiocyanate salt is present, for example. As a guideline, the A260/A230 is best if greater than 1.5.
Cell Lysis Buffer Recipe
| Reagent | Final Concentration | per 500 mL |
| 1 M Tris pH 8.0 | 10 mM | 5 mL |
| 5 M NaCl | 100 mM | 10 mL |
| 0.5 M EDTA pH 8.0 | 10 mM | 10 mL |
| 10% SDS | 0.50% | 25 mL |
| dH2O | to 500 mL |
- Add 20 µL of a 20 mg/mL Proteinase K per 1 mL of lysis buffer