What are the steps of DNA extraction?

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.

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.

1. Harvest cells from the culture vessel.
2. Pellet cells by centrifugation at 1000 x g for 1 minute and discard supernatant.
3. Resuspend the cell pellet in 100 µL cold PBS and mix by pipetting up and down repeatedly.
4. 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
5. 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.
6. 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.
7. Spin at max speed for 5 minutes, and carefully transfer the upper aqueous layer to a fresh Eppendorf tube.
8. To precipitate DNA add 1 mL of 100% EtOH (room temperature), close tube and gently invert until DNA precipitate forms.
9. Incubate the tube at room temperature for 15 – 30 minutes.
10. Spin at max speed for 5 minutes and carefully remove and discard supernatant.
11. Wash the DNA pellet with 1 mL 70% EtOH (-20°C) and invert several times.
12. Spin at max speed for 2 minutes, and carefully remove and discard supernatant.
13. Dry the DNA pellet at room temperature overnight or dry using a vacuum concentrator
14. 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.