Isolation of Badnavirus Particles from Infected Leaves

Аннотация

Source: Verchot, J., Thapa, A., 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 isolation and purification of Badnavirus particles from infected plant tissue using differential centrifugation and chemical lysis to prepare virus-enriched fractions for downstream genomic analysis.

Протокол

1. General Virus Purification by Differential Centrifugation Using Standard Method

1. First, cut 80-100 g of leaves from diseased plants and grind in a Waring blender at 4 °C using 200 mL grinding buffer 0.5 M disodium hydrogen phosphate (Na₂HPO₄), pH 7.2, and 0.5% (w/v) sodium sulfite (Na₂SO₃). Wear a laboratory coat and gloves for all steps of this procedure.
2. Then, transfer the homogenate (300 mL) to a 1.0 L beaker. Add 18 g of urea and 25 mL of 10% nonionic detergent to the homogenate inside a chemical hood.
   NOTE: For this step, it is best to wear safety goggles and a simple breathing mask for personal protection.
3. Stir with a magnetic stirrer briefly in the hood and cover the beaker with foil. Then transfer the foil-covered beaker to a cold room and stir with a magnetic stirrer overnight at 4 °C.
4. Transfer the homogenate to centrifuge rotor bottles (250 mL containers) and centrifuge in a fixed-angle rotor at 4,000 x g for 10 min at 4 °C. In a chemical fume hood, recover the supernatant and filter through 4 layers of cheesecloth.
5. Divide the homogenate among 38.5 mL polypropylene centrifuge tubes and centrifuge for 2.5 h at 40,000 x g at 4 °C. Typically, check for the presence of a green pellet at the bottom of the tube and a white pellet along the length of the tube. Pour off the supernatant and retain both pellets; place samples on ice.
   NOTE: The green pellet contains chloroplasts, starch, and other organelles.
6. Working in a chemical hood, use a rubber policeman to separate the pellets. Resuspend the white pellet in each rotor bottle in 1 mL of double-distilled water (ddH₂O) over the course of 1-2 h while maintaining the suspensions overnight at 4 °C to allow the materials to fully dissolve into solution. Centrifuge the suspension at 6,000 x g and at 4 °C for 10 min to remove the remaining debris.
7. Centrifuge the concentrated suspension at 136,000 x g for 2 h at 4 °C to pellet virions. Resuspend the pellets in 1 mL of buffer (50 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), pH 7.5, 5 mM magnesium chloride or MgCl₂).

Материалы

Список материалов, использованных в этой статье

Имя	Компания	Каталожный номер	Комментарии
NaH₂PO₄	Sigma-Aldrich St. Louis MO	S5976	Grinding buffer for virus purification
Na₂HPO₄	Sigma-Aldrich	S0751	Grinding buffer for virus purification
Na₂SO₃	Thermo-Fisher Waltham, MA	28790	Grinding buffer for virus purification
urea	Thermo-Fisher	PB169-212	Homogenate extraction
Triton X-100	Sigma-Aldrich	X-100	Homogenate extraction
Cheesecloth	VWR Radnor, PA	21910-107	Filter homogenate
Tris	Thermo-Fisher	BP152-5	Pellet resuspension& DNA resuspension buffers
MgCl₂	Spectrum, Gardena, CA	M1035	Pellet resuspension buffer
EDTA	Spectrum	E1045	Stops enzyme reactions
Proteinase K	Thermo-Fisher	25530	DNA resuspension buffer
Phenol:chloroform:isoamylalcohol	Sigma-Aldrich	P2069	Dissolve virion proteins
DNAse I	Promega	M6101	Degrade cellular DNA from extracts
95% ethanol	Sigma-Aldrich	6B-100	Virus DNA precipitation
Laboratory blender	VWR	58984-030	Grind leaf samples
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
Magnetic stir plate	VWR	75876-022	Mixing urea into samples overnight
Rubber policeman	VWR	470104-462	Dissolve virus pellet