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 .
