Purification of DNA from Virions Using Organic Solvents

الملخص

Source: Verchot, J., et al. Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes. J. Vis. Exp. (2018)

This video demonstrates the organic solvent-based extraction and purification of viral DNA from virions. It shows how DNA is separated from proteins and lipids using phenol-chloroform extraction, followed by the precipitation using ethanol. The extracted DNA is purified by gel filtration for downstream applications.

البروتوكول

1. General Virus Purification by Differential Centrifugation Using Standard Method

1. Disrupt virions with 40µL of 2µg/µL proteinase K at 37°C for 15min.
2. Work inside a chemical hood to recover virion DNA by organic extraction. Wear a face shield, gloves, and a laboratory coat during the extraction for protection against potential acute health effects. Add 1 volume of phenol-chloroform-isoamyl alcohol (49:50:1) to the sample and shake by hand for 20s. Centrifuge at room temperature for 5 min at 16,000 xg. Remove the upper aqueous phase and transfer to a new tube. Repeat this extraction two or more times. Dispose of the organic phase by placing it in a glass waste bottle for proper institutional chemical disposal.
3. Concentrate the DNA using ethanol precipitation. Use 0.3M final concentration of sodium acetate (pH 5.2) and 2.5 volumes of 95% ethanol. Place samples at -20°C for 30-60 min and centrifuge at 13,000 xg for 10-20 min to pellet the DNA.
4. Working at a laboratory bench, resuspend the DNA pellet in 1mL of 0.1mM TE [Tris-EDTA(Ethylene diamine tetraacetic acid)] buffer (pH 8.0). Filter the suspension through a commercial gel filtration column (normally used for polymerase chain reaction (PCR) clean up) to eliminate salts and low molecular weight material that might impede NGS (next generation sequencing).

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
Tris	Thermo-Fisher	BP152-5	Pellet resuspension& DNA resuspension buffers
MgCl₂ (Magnesium Chloride)	Spectrum, Gardena, CA	M1035	Pellet resuspension buffer
EDTA (Ethylenediaminetetraacetic acid)	Spectrum	E1045	Stops enzyme reactions
Proteinase K	Thermo-Fisher	25530	DNA resuspension buffer
Phenol:chloroform:isoamylalcohol	Sigma-Aldrich	P2069	Dissolve virion proteins
95% Ethanol	Sigma-Aldrich	6B-100	Virus DNA precipitation
Floor model ultracentrifuge &Ti70 rotor	Beckman Coulter, Irving TX	A94471	Separation of cellular extracts
Floor model centrifuge and JA-14 rotor	Beckman Coulter	369001	Separation of cellular extracts
PCR clean-up kit	Promega	A9281	Prepare PCR products for cloning
DNAse I	Promega	M6101	Degrade cellular DNA from extracts