Name: preparation of viral dna from nucleocapsids
Uploaded: 2011-08-16T12:00:00
Description: Watch this Scientific Journal Video about Preparation of Viral DNA from Nucleocapsids at JoVE.com

लेखक ग्रेग स्मिथ, लीज़ा Pomeranz, मैट Lyman, MARLIES Eldridge, Halina Staniszewska Goraczniak, और ठीक ट्यूनिंग इस प्रोटोकॉल में Enquist प्रयोगशाला के सदस्यों के योगदान की सराहना करते हैं.

1. प्रथम दिवस: वायरल संक्रमण और Buffers की तैयारी <ol><li> ऊतक संस्कृति कोशिकाओं के संक्रमण के लिए 5-10 (15 सेमी व्यास) व्यंजन तैयार करने के लिए, pseudorabies (PRV) वायरस या दाद के लिए वेरो कोशिकाओं के लिए जैसे पीके 15 कोशिकाओं सिंप?...

<ol>
	<li>Szpara, M. L., Parsons, L., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+variability+in+clinical+and+laboratory+isolates+of+herpes+simplex+virus+1+reveals+new+mutations.">Sequence variability in clinical and laboratory isolates of herpes simplex virus 1 reveals new mutations.</a> J Virol. 84, 5303-5313 (2010).</li><li>Banfield, B. W., Kaufman, J. D., Randall, J. A., Pickard, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+pseudorabies+virus+strains+expressing+red+fluorescent+proteins:+new+tools+for+multisynaptic+labeling+applications.">Development of pseudorabies virus strains expressing red fluorescent proteins: new tools for multisynaptic labeling applications.</a> J Virol. 77, 10106-10112 (2003).</li><li>Kobiler, O., Lipman, Y., Therkelsen, K., Daubechies, I., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpesviruses+carrying+a+Brainbow+cassette+reveal+replication+and+expression+of+limited+numbers+of+incoming+genomes.">Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes.</a> Nat Commun. 1, 146-146 (2010).</li><li>Enquist, L. W., Madden, M. J., Schiop-Stanley, P., Vande Woude, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+herpes+simplex+type+1+DNA+fragments+in+a+bacteriophage+lambda+vector.">Cloning of herpes simplex type 1 DNA fragments in a bacteriophage lambda vector.</a> Science. 203, 541-544 (1979).</li><li>Smith, G. A., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+transposon+mutagenesis+in+Escherichia+coli+of+a+full-length+infectious+clone+of+pseudorabies+virus,+an+alphaherpesvirus.">Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus.</a> J Virol. 73, 6405-6414 (1999).</li><li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of virology. , (2008).</li><li>Pomeranz, L. E., Reynolds, A. E., Hengartner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pseudorabies+virus:+impact+on+neurovirology+and+veterinary+medicine.">Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine.</a> Microbiol Mol Biol Rev. 69, 462-500 (2005).</li><li>Roizman, B., Pellett, P. E., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology. , 2381-2397 (2001).</li><li>Mendez, I. I., Hermann, L. L., Hazelton, P. R., Coombs, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+analysis+of+freon+substitutes+in+the+purification+of+reovirus+and+calicivirus.">A comparative analysis of freon substitutes in the purification of reovirus and calicivirus.</a> J Virol Methods. 90, 59-67 (2000).</li><li>Gharabaghi, F., Aymard, M., Trotemann, P., Gerdil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+simplified+micromethod+for+subtyping+varicella-zoster+virus.">A rapid and simplified micromethod for subtyping varicella-zoster virus.</a> J Med Virol. 31, 129-134 (1990).</li><li>Granstedt, A. E., Szpara, M. L., Kuhn, B., Wang, S. S., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+monitoring+of+in+vivo+neural+activity+using+a+circuit-tracing+pseudorabies+virus.">Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus.</a> PLoS One. 4, e6923-e6923 (2009).</li></ol>

इस प्रोटोकॉल के भाग मूल BSL4 परिस्थितियों में वायरल डीएनए अलगाव के लिए विकसित किए गए, लेकिन यह गैर BSL स्थितियों के लिए समान रूप से अच्छी तरह से adapts 4 हम आमतौर पर इस प्रोटोकॉल का उपयोग करने के लिए अल्फा herpesviru...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियाँ </td></tr><tr><td> Freon (1,1,2-trichloro एक ,2,2 trifluoroethane) </td><td> मछुआ </td><td> T178 - 4 </td><td> के उचित निपटान पर दिशानिर्देशों Freon युक्त अपशिष्ट के लिए अपनी संस्था के साथ की जाँच करें, या मेंडेज़ एट अल देख. संभावित Freon 9. विकल्प के लिए </td></tr><tr><td> चरण ताला जेल ट्यूबों, भारी 15ml क्षमता </td><td> 5 प्रधान </td><td> 2302850 </td><td> ऐच्छिक </td></tr><tr><td> Polyallomer ultracentrifuge ट्यूबों </td><td> Beckman * </td><td> 331372 * </td><td> का चयन करें * अपने खुद ultracentrifuge के लिए उपयुक्त ट्यूबों, ये केवल एक उदाहरण के रूप में शामिल हैं </td></tr><tr><td> NP-40 / IGEPAL </td><td> सिग्मा </td><td> मैं-3021 </td><td></td></tr><tr><td> पी 15 कक्षों </td><td> ATCC </td><td> सीसीएल-33 </td><td></td></tr><tr><td> वेरो कोशिकाओं </td><td> ATCC </td><td> सीसीएल-81 </td><td></td></tr><tr><td> पीबीएस </td><td> HyClone </td><td> SH30028.03 </td><td></td></tr></tbody></table>

preparation of viral dna from nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

Watch this Scientific Journal Video about Preparation of Viral DNA from Nucleocapsids at JoVE.com

Preparation of Viral DNA from Nucleocapsids

हम संक्रमित कोशिकाओं से उच्च शुद्धता herpesvirus nucleocapsid डीएनए अलग करने की प्रक्रिया का वर्णन. अंतिम समाधान से कब्जा कर लिया डीएनए उच्च एकाग्रता और पवित्रता है, यह आदर्श उच्च throughput अनुक्रमण, उच्च विश्वस्तता पीसीआर प्रतिक्रियाओं, और transfections के लिए नए वायरल recombinants उत्पादन के लिए उपयुक्त बना.

वायरल डीएनए के Nucleocapsids से तैयार

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

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