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

作者欣赏，莉萨彭慕兰格雷格史密斯，马特 - 莱​​曼，Marlies埃尔德里奇，Halina Staniszewska Goraczniak和Enquist实验室成员在本议定书微调的贡献。

1。第一天：病毒感染和缓冲区的制备<ol><li>准备5-10菜的组织培养细胞感染（直径15厘米），如PK - 15细胞为伪狂犬病病毒（PRV）Vero细胞或疱疹病毒（HSV）单纯。 </li><li>当细胞是95 - 100％融合，感染（MOI），5-10。要做到这一点，接种使用病毒库存总量在4毫升每盘每盘，然后孵育1小时板块在37 ° C。岩石板轻轻每15分钟一班，以确保细胞单层仍然完全由涂病毒接种。同时，热情为下一步的介质。 </li...

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	<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>

本协议的某些部分最初是病毒DNA隔离BSL4条件，但它同样适应非BSL条件。4，我们通常使用此协议隔离α-疱疹病毒，伪狂犬病毒和HSV - 1，其中有DNA的基因组DNA封闭在一个蛋白质衣壳和脂质信封包围。7,8然而，它可能是直接适应其他大型DNA病毒，包括β-和γ-疱疹病毒和腺病毒。类似的提取通常用于RNA病毒以及9。 这个DNA的制备，净化多种方法，包括可能的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>氟里昂（1,1,2 - 三氯-2,2,2 - 三氟） </td><td>费舍尔</td><td> T178 - 4 </td><td>请与您的机构适当处置的“指引”氟利昂含有废物，或者看到门德斯等。 9潜在的氟利昂替代品。 </td></tr><tr><td>锁相凝胶管，重型15毫升能力</td><td> 5 PRIME </td><td> 2302850 </td><td>可选</td></tr><tr><td> Polyallomer超速离心机管</td><td>贝克曼* </td><td> 331372 * </td><td> *选择管适合你自己的超速离心机，这些都是仅作为一个例子</td></tr><tr><td> NP - 40 / IGEPAL </td><td>西格玛</td><td>我- 3021 </td><td></td></tr><tr><td> PK - 15细胞</td><td> ATCC </td><td> CCL - 33 </td><td></td></tr><tr><td> Vero细胞</td><td> ATCC </td><td> CCL - 81 </td><td></td></tr><tr><td> PBS </td><td> Hyclone公司</td><td> SH30028.03 </td><td></td></tr></tbody></table>

preparation of viral dna from nucleocapsids

病毒是专性细胞寄生虫，因此它们的DNA的研究需要从宿主细胞中的污染物和DNA分离病毒的材料。几个下游应用需要大量的纯病毒DNA，这是由本议定书规定。这些应用程序包括病毒基因组测序，去除宿主DNA病毒序列数据输出是至关重要的优化，新的重组病毒株生产的，共转染纯化的质粒和线性病毒DNA有利于重组 。1,2， 3 此过程采用的提取和基于密度离心分离纯化线性疱疹病毒感染的细胞的核衣壳的DNA相结合 。4,5初步纯化步骤的目的是分离纯化病毒衣壳，其中包含和保护过程中的病毒DNA的提取和离心步骤去除细胞的蛋白质和DNA。核衣壳裂解，然后释放病毒DNA，最后两个酚 - 氯仿的步骤删除剩余的蛋白质。最后的DNA从溶液中捕获的是高度集中和纯粹，平均外径为 1.90 260/280。根据感染的细胞，病毒DNA范围从150-800微克或以上的产量的数量。这种DNA的纯度，使之稳定，在长期储存在4C。这种DNA，因此非常适合于高通量测序，高保真PCR反应，并转染。 开始前的协议，重要的是要知道每道菜的细胞（如平均15厘米在一个融合的菜，8 × 10 6 PK - 15细胞），和滴度的病毒的股票要使用的平均数（如1 × 10 8每毫升空斑形成单位）。这些是必要的适当的感染复数（MOI）计算的协议。， 例如 6，感染15 PK - 15细胞与上述病毒的股票在MOI为5厘米的菜，你会使用400μL病毒的股票和稀介质为3.6毫升（共接种4毫升，一个15厘米板卷）。 多个病毒DNA的制剂可以在同一时间准备。同时筹备工作的数量是有限的，只有超速离心机转子举行管的数量（每病毒之一;见下面的步骤3.9）。在这里，我们描述了，就像做一个病毒程序。

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

Preparation of Viral DNA from Nucleocapsids

我们描述了从被感染的细胞中分离高纯度的疱疹病毒衣壳DNA的过程。从溶液中捕获的是最后的DNA浓度和纯度高，使得它非常适合高通量测序，高保真PCR反应，并产生新的病毒重组体转染。

从核衣壳的病毒DNA的制备

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

Research

JoVE Journal

Immunology and Infection

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质粒DNA重组流感病毒的一代

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一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

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建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

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建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

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