Hewson Lab at Cornell

Team Aquatic Virus - Microbial Oceanography, P.I. Ian Hewson, Ph.D.

Preparation of DNA and RNA Viriomes

Filter Chloroform Nuclease Virus Purification Protocol

Terry Fei Fan Ng and Mya Breitbart
Modified for the Hewson Lab by Julie Brown
Elements of protocol adapted from “Thurber et al., Laboratory procedures to generate viral metagenomes, Nature Protocols, 2009”

  1. If working with Animal Samples:
    1. Homogenize in sterile SM buffer, or sterile PBS buffer using a mortar and pestle.
    2. Centrifuge homogenate at 10,000xg at 4° C for 10 minutes to remove host tissue.
  2. Filter supernatant (or sample if beginning with a virus concentrate or water sample) through a 0.2 µm anodisc or sterivex filter into a new, sterile oak ridge tube, with a maxiumum of 35 ml of filtrate per tube.
    1. If working with freshwater, add NaCl to a final concentration of 1M
    2. Add 10% by weight PEG 8000. 
      1. For 35 ml of filtrate add 3.5 grams of PEG.
  3. Precipitate samples overnight at 4° C.
  4. Spin samples at 15,000*g for 30 minutes, pour off supernatant and carefully remove excess liquid with a micropipettor.
  5. Resuspend pellet in 1 ml virus-free PBS or water.
    1. These samples are predominantly virus concentrate and may be stored in the -80C.
    2. Do not re-suspend in a solution containing EDTA. 
    3. If you are feeling extra careful, filter 1 ml sample again through a 0.2µm filter.
  6. Treat entire sample or a sub-sample with 0.2 volumes chloroform, flick to mix, and incubate at room temperature for 10 minutes.
    1. If you are extracting from the entire 1 ml, this means that you should add 200 µl chloroform.
    2. Note that residual PEG may cause a white layer to form between Chloroform and aqueous layers.
    3. Spin briefly after ten minutes to separate layers.
  7. Transfer aqueous layer to a new eppendorf tube.
  8. Incubate sample with 2.5U DNase I, and 0.25U RNase at 37 degrees C for 3 hours.
    1. DNase I generally comes as 1U per µl, check your solution to make sure this is the case.
  9. Add 0.2 volumes of 100mM EDTA to your sample for a final concentration of 20mM EDTA.
    1. If you are using 0.5M EDTA, add .04 volumes EDTA.

You now have pure viruses.

DNA and RNA may now be extracted using ZR viral extraction kits (Zymo Research), or any other method of choice.

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