Preparation of Virus DNA from Seawater for Metagenomics
Precipitate 0.2µm filtered seawater with iron chloride and store the filters at 4°C .
Resuspend the precipitated virus in Mg - EDTA - Ascorbate ( or Oxalate ) buffer just prior to DNA extraction ( also see Ferric Chloride Resuspension Buffer protocol ) .
Treat the resuspended virus preparation with DNase I for 2 hours at room temperature .
Inactivate the enzyme with EDTA and EGTA .
Concentrate the resuspended virus preparation using centrifugal concentrators down to 3 - 5 ml total volume if performing CsCl cleanup or to 1 - 2 ml total volume if proceeding directly to DNA extraction .
Remove the concentrate to a fresh tube .
Rinse the membrane with an additional 0.5 - 1 ml resuspension buffer by pipetting up and down along the membrane .
Pool with the concentrated sample .
At this point , either layer this onto premade CsCl gradients and isolate the virus fraction according to the protocol Cesium Chloride Purification of Viruses ( or alternatively , Cesium Chloride DNA Extraction of Viruses using Wizard Prep Columns ) or proceed directly to Wizard Prep purification of DNA .
Extract the DNA using Wizard Prep columns and resin Calculate the amount of DNA recovered using Quant - iT dsDNA Pico Green assay kit following manufacturer’s directions
