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[DESCRIPTION]
OptiPrep gradients, as described for purifying viral isolates, may also be used for purifying marine viral assemblages. There are as yet, however, few descriptions of this application in the literature. CsCl gradients, on the other hand, have been used extensively for purifications of viral isolates and natural viral assemblages. Given the continued popularity of CsCl gradients, and their well-characterized performance, we present the following two-part purification and fractionation protocol for viruses in CsCl gradients. The first part involves relatively quick sedimentation through a step-gradient to remove the bulk of the contaminants. This is followed by a higher resolution continuous gradient to separate the viruses from residual contaminants and to separate viruses having differing buoyant densities from one another. We assume the starting material for the following procedure to be a concentrated suspension of viruses (whether filtered to remove cells or not).

[GUIDELINES]
<strong>Materials and Reagents</strong><br /><br /> • Ultracentrifuge <br />• Swinging-bucket rotor (Beckman Coulter SW 41 Ti or Sorvall TH-641) <br />• Polyallomer tubes (Beckman Coulter part No. 331372; Sorvall part No. 03669) <br />• CsCl (molecular biology grade) <br />• SM Buffer (100 mM NaCl, 8 mM MgSO<sub>4</sub>, 50 mM Tris pH 7.5)<br /><br /><strong>Discussion</strong><br /> <br />The point of the step gradient in this two-step protocol is to achieve a quick initial purification of viruses, which can then be purified to greater degree in the subsequent continuous gradient. The spin is kept short to minimize diffusion between layers, which would eventually result in a continuous gradient. Because the spin is short, not all material may reach its equilibrium position in the gradient. Viruses, having relatively high sedimentation coefficients, reach their equilibrium positions more quickly than many other dissolved macromolecules. Since all the material starts at the top of the gradient, viruses can therefore be most efficiently separated from less dense material (e.g., lipids, proteins, and most bacteria) as well as molecules that are more dense, but which have low sedimentation rates (small pieces of DNA or RNA). The subsequent continuous gradient, when centrifuged to equilibrium, provides good separation of viruses from contaminants and different viruses from one another. Since the less dense contaminants are mostly removed in the step gradient, which is unloaded from the bottom, we recommend unloading the subsequent continuous gradient from the top, thereby minimizing contamination from free nucleic acids, which will either band below the viruses (DNA) or form a pellet at the bottom of the tube (RNA). Viruses have been purified for metagenomic analyses using only a step gradient (Angly et al. 2006; Breitbart et al. 2004; Breitbart et al. 2002; Vega Thurber et al. 2009), or no gradient at all (Bench et al. 2007; Helton and Wommack 2009). In the latter cases 0.2-µm filtration and nuclease digestion are relied upon to remove nonviral nucleic acids. Since many virus genes recovered from the environment may not be recognized as viral based only on their sequence (Edwards and Rohwer 2005), having a highly purified virus preparation increases one’s confidence that any novel sequences recovered do indeed derive from viruses rather than from cellular life forms. For that reason, one may find the complete two-part gradient purification protocol especially desirable for viral metagenomic studies. A rigorous gradient purification would be particularly important if the sample had not been first filtered to remove prokaryotes. <br /><br />Fixed-angle rotors (including vertical, near-vertical, and others) are commonly used for equilibrium buoyant density gradients, especially self-forming gradients, because the centrifugation times required to approach the equilibrium gradient shape can be much shorter. However, the use of a swinging bucket rotor for the continuous gradient, as presented here, has several advantages: 1) only a single rotor is needed for the both the step and the continuous gradients, 2) the open-top tube simplifies gradient unloading from the top, and 3) there is less chance of contaminating the viruses with dissolved nucleic acids or cellular material. Any contaminating nucleic acid, especially RNA, that pellets during the run will be located at the very bottom of the tube where it will not be in contact with the virus bands. Material that is less dense than the least dense portion of the gradient will not pellet, but will float at the top of the gradient. In any type of fixed angle rotor, these potential contaminants will pellet to some degree on the sides of the tube and come in contact with the viral bands during deceleration as the gradient reorients in the tube. This material could contaminate the recovered fractions if it is dislodged or diffuses from the pellets during unloading. This is of particular concern for a sample that has not been first purified through a step gradient. <br /><br />To partially compensate for the longer centrifugation times required for gradient to reach equilibrium in a swinging bucket rotor, we present a protocol in which a continuous gradient forms by diffusion from an initial step gradient. The modest amount of extra effort needed to prepare the step gradient is compensated by a significantly shorter run time. If one were to start with a homogeneous CsCl solution in an SW 41 or TH-1641 rotor, it could take ≥ 80 h for the gradient, and the constituents within it, to approach equilibrium. Another advantage of the step gradient is that it provides a dense cushion at the bottom of the tube that prevents viruses from pelleting early in the run before the gradient has fully formed.  <br /><br /><strong>WARNING!</strong> The protocol we present here is tailored to the specified rotors and operating conditions. Many other rotors and centrifugation conditions could be used instead. However, if you wish to change the conditions or adapt the methods to a different rotor, it is critical that you ensure that the new conditions are within the safe limits for centrifugation of CsCl gradients. Centrifugation of CsCl solutions at certain combinations of concentration, temperature, and rotor speed can result in CsCl crystallization at the bottom of the tube. The high density of the crystals will exceed the tolerance of the rotor and could result in catastrophic rotor failure.

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