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[DESCRIPTION]
This protocol is a minor modification of that reported by Culley and Steward (2007). As the starting point for this protocol, we assume that viruses have been collected on an aluminum oxide 0.02-µm syringe-tip filter (Anotop, Whatman), but other filters capable of capturing viruses may be substituted.<br /><br />This is a protocol from: <br /><br />Steward, G. F. and A. I. Culley. 2010. Chapter 16: Extraction and purification of nucleic acids from viruses. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_154.pdf" target="_blank">published manuscript</a> for additional information.

[GUIDELINES]
<strong>Materials and equipment:</strong> <br />• Total nucleic acid extraction kit (MasterPure, Epicenter) <br />• Optional: polyacryl carrier (Molecular Research Center)<br /> • Syringes (sterile, disposable, with Luer-Lok tips) <br />• Luer-Lok female–female adapter fittings <br />• Hybridization oven with rotisserie<br /><br /><strong>Assessment:</strong> One caveat in extracting from aluminum oxide membrane filters is that they can irreversibly bind DNA under certain conditions (Dames et al. 2006). In particular, guanidinium-containing extraction buffers facilitate the binding of DNA to aluminum oxide (Gerdes et al. 2001) and are likely to result in low yields from the filters. For this reason, we do not recommend extracting from Anotop filters using the lysis buffers from any of the popular silica column–based kits. The Gentra PureGene Kit (Qiagen) is similar to the MasterPure kit and may work as well. These latter kits are based on a published protocol (Miller et al. 1988) that could be adapted for extraction from a filter. A version of that protocol designed for simultaneous DNA and RNA extraction (Yu and Mohn 1999) could also be used by employing heat (65°C, 15 min) instead of bead beating to facilitate lysis. It is worth noting that SDS and phosphate buffer appear to inhibit the binding of nucleic acid to aluminum oxide (Gerdes et al. 2001; Dames et al. 2006) and should aid in recovery. If one wishes to recover RNA using a self-made recipe, then we would recommend including an RNAse inhibitor (e.g., RNASecure, Ambion) in the extraction buffer. <br />     We have recovered both viral RNA and DNA suitable for PCR amplification from aluminum oxide filters using essentially the procedure as described above (Culley and Steward 2007; Culley et al. 2008). A modification added here is the introduction and removal of the extraction buffer in a direction counter to that of filtration during sample collection (i.e., backflushing). According to the Anotop specifications, these filters are not designed to be backflushed, or to be operated at temperatures above 40°C. We have found that the filter can rupture if too much pressure is applied during backflushing, especially after incubating at 65°C. Injecting the extraction buffer slowly and removing the extract by gentle aspiration seem to avoid this problem. Although we have not tested the protocol described here on other filter types, the procedure should work as well for any direct flow filter capsule capable of retaining viruses.<br /><br />

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