Iron Chloride Precipitation of Viruses from Seawater
Assemble two 142mm filtering towers and attach the filter apparatus to a peristaltic pump with a pressure gauge (maximum pressure = 15 psi).
Pre-filter 20L of seawater using a 150mm Whatman GF/A filter.
Filter again to remove bacteria using a 0.22um, 142mm Millipore Express Plus filter.
Treat the virus fraction (0.22μm filtrate) with FeCl3 to precipitate the viruses by adding 1ml of 10g/L Fe Stock Solution to each 20L of filtrate.
Shake vigorously for 1 minute.
Add an additional 1ml of 10g/L Fe stock solution to each 20L of filtrate (for a total of 2ml Fe Stock Solution per 20L filtrate).
Shake vigorously for 1 minute.
Let the FeCl3 treated filtrate sit for 1 hour at room temperature.
Filter the FeCl3 treated filtrate using a 1.0μm, 142mm, polycarbonate (PC) membrane filter on top of a 0.8μm, 142mm, support filter and attaching the filter apparatus to a peristaltic pump with a pressure gauge (maximum pressure = 15 psi).
Place all of the polycarbonate filters from the 20L into a 50ml centrifuge tube being careful not to scrape off any of the FeCl3 on the edge of the tube (having precipitate facing out aids in dissolving the precipitate).
Discard the Supor support filter.
Prepare fresh 0.1M EDTA; 0.2M MgCl2; 0.2M Ascorbate Buffer, pH6.5.
Add 1ml buffer for each 1mg Fe, which is 20ml of 1x buffer for the 20L seawater precipitate (10ml of a 2X concentrated buffer).
Shake the tube vigorously.
Place tubes on a rotator and rotate at 4°C overnight.
Leave stored at 4°C until ready to analyze or further purify the viral fraction.
To collect the resuspended precipitate, open the tube and collect liquid into a fresh tube.
Using a sterile pipet, applicator stick, or bleached and rinsed forceps pull an edge of the filter onto the tube edge so it is caught in the lid when secured.
Centrifuge at low speed (less than 1000 rpm) for 3-5 minutes to recover more buffer.
If the filter has precipitate on it, add more buffer, incubate again for a couple hours, and collect liquid as stated above.
