Purification of DNA from Virions Using Organic Solvents

Start with extracted viral particles or virions, where the viral DNA is enclosed by capsid proteins.

Add Proteinase K and incubate to digest the proteins around viral DNA.

Add an organic solvent mixture of phenol, chloroform, and isoamyl alcohol. Shake and centrifuge.

Water-soluble DNA moves to the upper layer. Phenol-chloroform denatures proteins and helps separate lipids, while isoamyl alcohol promotes clear phase separation.

Transfer the aqueous layer to a new tube. Add sodium acetate and ethanol.

Incubate in cold conditions. Sodium acetate shields negative charges on the DNA while ethanol reduces its water solubility to precipitate it.

Centrifuge to pellet the DNA. Remove the supernatant.

Resuspend the pellet in the Tris-EDTA buffer for stabilizing the DNA.

Load the DNA solution onto a gel filtration column and centrifuge.

Large DNA molecules pass through the column, while small molecules are retained within the porous gel matrix, yielding purified DNA for downstream applications.

Begin this procedure by disrupting the virions. Add forty microliters of a two microgram per microliter proteinase K solution to the pellet suspensions and incubate at 37 degrees Celsius for 15 minutes. Add one