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[DESCRIPTION]
This protocol describes the use of a sucrose cushion to purify viruses. It is the&nbsp;Matthew Sullivan Lab adaptation of the Shannon Williamson protocol.<br /><br />DNase I treatment, CsCl purification, and&nbsp;sucrose purification methods were compared using replicated viral metagenomics in Hurwitz et al. 2012.<br /><br /><strong>Citation:</strong> Hurwitz, B. L., Deng, L., Poulos, B. T., &amp; Sullivan, M. B. (2012).<a href="http://www.ncbi.nlm.nih.gov/pubmed/22845467" target="_blank"> Evaluation of methods to concentrate and purify ocean virus communities through comparative, replicated metagenomics</a>. Environmental Microbiology. doi:10.1111/j.1462-2920.2012.02836.x

[GUIDELINES]
<strong>Shannon Williamson’s Protocol</strong>   <br /><br />1.)  DNase I Treatment (Can also be done post sucrose cushion) <br /><br />     1.1  Dilute the sample to approximately 10-­-20% glycerol with MilliQ water. Most samples will be in 50% glycerol solutions.  This will generally be a volume of ~20ml.  <em>Only applies if glycerol was used as a cryo-­-protectant.  </em> <br /><br />     1.2  Add 1 unit of DNase I (RNase free) for every milliliter of sample.   <br /><br />     1.3  Mix sample and incubate at room temperature for 2 hours.   <br /><br />     1.4  Stop reaction by adding EDTA and EGTA to a final concentration of 100mM.   <br /><br />     1.5  Store at 4°C  <br /><br /><br /><strong>2.)  Pelleting Viral Particles Through a Sucrose Cushion</strong> <br /><br />     2.1  Rinse one ultracentrifuge tube per sample with sterile water.   <br /><br />     2.2  Load sample (no more than ~25 to 30ml) into ultracentrifuge tube.   <br /><br />     2.3  Add up to 10ml of 38% Sucrose in SM buffer to the bottom of ultracentrifuge tube to form a sucrose cushion. The tube should be filled to the very top and balanced to within  +/- 0.1g to avoid being crushed by the ultracentrifuge’s vacuum.   <br /><br />     2.4  Centrifuge for 3 hours, 23°C at 32,000 RPM in a SW32.1Ti rotor. See <strong>Note</strong> below.   <br /><br />     2.5  Gently pipette off the supernatant and save at 4°C.   <br /><br />     2.6  Allow pellet to dry for 5 - 10 minutes, and resuspend in 500ul of TE.   <br /><br />     2.7  Add EGTA and EDTA to a final concentration of 100mM. <br /><br /><strong>Note:  Email correspondence with author indicated that the ultracentrifugation is at 32,000 RPM as opposed to 32,000 x g.  The difference is very large between these two:  in this rotor 32,000 x g would be about 13,000 RPM whereas 32,000 RPM = 180,000 x g. See email correspondence below.  BTP  </strong>     <br /><br /><br /><strong>Emails concerning protocol details:</strong>  <br /><br />From: Matthew Sullivan  <br />Sent: Monday, October 19, 2009 3:32 PM<br />To: Williamson, Shannon<br />Subject: few quick Qs <br /><br />Hey Shannon,<br />We're setting up to implement your sucrose protocol. A few questions came up ...  <br /><br />1) Should the EDTA and EGTA both be at 100mM final concentration or is this a combined molarity of both?  <br /><br />2) Why are EDTA and EGTA also added to the resuspended pellet? (is DNase still potentially present)  <br /><br />3) Your protocol says to use a SW32.1Ti rotor and just prior says to use no more than 25-­30ml of sample with 10ml sucrose but the tubes for this rotor only holds 17ml (whereas the SW32Ti rotor uses 38ml tubes).  Can you clarify the amounts and rotor?  (just want to confirm g force for the centrifugation since 32K rpm in 32.1Ti = 187,000g  whereas 32K RPM in 32Ti = 175,000g)<br /><br /> From: "Williamson, Shannon"  <br />Date: October 21, 2009 9:33:34 AM GMT-­-07:00<br />To: "Matthew Sullivan"<br />Subject: RE: few quick Qs <br /><br />Hi Matt, Here are some answers for you...  <br /><br />1) 100mM is the final concentration of each. So 100mM EGTA and 100mM EDTA.  <br /><br />2) The EDTA/EGTA is added here just as a precaution to remove Mg and Ca in the case that we have any enzymes after we do the DNA extraction. The possibility of having bacterial cells here is very slim but we do not take chances especially with viral DNA yields being what they are. Also, it is possible to do the DNAse reaction after the sucrose cushion, which is recommended if you want to work in smaller volumes and save money and reagents.  <br /><br />3) For the sucrose cushion you want to make sure you have about 1/8-1/6 of the total ultra centrifuge tube volume to be your sucrose cushion with the remainder being your concentrated VC. As long as you can get up to 30,000g or 32,000g it doesn't matter which rotor or tubes you use. It really comes down to how comfortable you are concentrating your VC to small enough volumes to fit the tubes.  We have done the sucrose cushion in the 5ml, 15ml, and 38ml ultra tubes, and all have worked well.    <br /><br />Please let me know if you have any additional questions. <br /><br />Best wishes,<br />Shannon <br /><br />From: Matthew Sullivan<br />Sent: Thursday, October 22, 2009 9:31 AM<br />To: Williamson, Shannon<br />Subject: Re: few quick Qs <br /><br />Hey Shannon,<br /><br />Thanks so much for the response ... do you mean 30,000 g or RPM below?   (my first time doing a sucrose gradient and I'm told these things are   very spin sensitive)<br /><br />All the best,<br />Matt <br /><br />From: "Williamson, Shannon"<br />Date: October 22, 2009 1:35:45 PM GMTÂÂ07:00<br />To: "Matthew Sullivan"  <br />Subject: RE: few quick Qs <br /><br />It's RPM, sorry about that!<br /><br />From: Matthew Sullivan  <br />To: Bonnie Poulos  <br />Subject: Fwd: few quick Qs<br />Date: Thu, 22 Oct 2009 13:37:54 ÂÂ0700 <br /><br />Hey Bonnie it's RPM ...

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