Name: extraction of high molecular weight dna from microbial mats
Uploaded: 2011-07-07T12:03:00
Description: Watch this Scientific Journal Video about Extraction of High Molecular Weight DNA from Microbial Mats at JoVE.com

这项工作是由美国国家科学基金会环境基因组计划（批准号：EF - 0723707）。

1。微生物细胞提取： <ol><li>彻底混合，均质，用无菌磨杵的微生物席。放置约30克（湿重）到无菌容器华林搅拌器匀浆垫材料约100毫升1 M氯化钠（或样品在使用特定的浓度），添加，并混合1分钟，以中等速度的三倍在-20℃冷冻1分钟的间歇冷却。转移到250 mL离心瓶的水泥浆和1 M氯化钠填补其余的空量。 NaCl溶液中，并准备在灭菌去离子水过滤消毒。 </li><li>进一步逐出晃动在室温下用30分钟vortexer?...

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	<li>Decho, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilms+in+intertidal+systems:+an+overview.">Microbial biofilms in intertidal systems: an overview.</a> Cont. Shelf Res. 20, 1257-1273 (2000).</li><li>Dupraz, C., Visscher, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+lithification+in+marine+stromatolites+and+hypersaline+mats.">Microbial lithification in marine stromatolites and hypersaline mats.</a> Trends Microbiol. 13, 429-438 (2005).</li><li>Steffan, R. J., Goksoyr, J., Boj, A. K., Atlas, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+DNA+from+soils+and+sediments.">Recovery of DNA from soils and sediments.</a> Appl. Environ. Microbiol. 54, 2908-2915 (1988).</li><li>Lee, Y. K., Kim, H. W., Liu, C. L., Lee, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+DNA+extraction+from+marine+bacteria+that+produce+extracellular+materials.">A simple method for DNA extraction from marine bacteria that produce extracellular materials.</a> J. Microbiol. Methods. 52, 245-250 (2003).</li><li>Roose-Amsaleg, C. L., Garnier-Sillam, E., Harry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+Purification+of+Microbial+DNA+from+Soil+and+Sediment+Samples.">Extraction and Purification of Microbial DNA from Soil and Sediment Samples.</a> Appl. Soil Ecol. 18, 47-60 (2001).</li><li>de Lipthay, J. R., Enzinger, C., Johnsen, K., Aamand, J., S&#248;rensen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+DNA+extraction+method+on+bacterial+community+composition+measured+by+denaturing+gradient+gel+electrophoresis.">Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis.</a> Soil Biol. Biochem. 36, 1607-1614 (2004).</li><li>Bey, B. S., Fichot, E. B., Dayama, G., Decho, A. W., Norman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+High+Molecular+Weight+DNA+from+Microbial+Mats.">Extraction of High Molecular Weight DNA from Microbial Mats.</a> Biotechniques. 49, 631-640 (2010).</li><li>Kakirde, S. K., Parsley, L. C., Liles, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+does+matter:+Application-driven+approaches+for+soil+metagenomics.">Size does matter: Application-driven approaches for soil metagenomics.</a> Soil Biology &#38; Biochemistry. 42, 1911-1923 (2010).</li><li>Rodon, M. R., August, P. R., Bettermann, A. D., Brady, S. F., Grossman, T. H., Liles, M. R., Loiacono, K. A., Lynch, B. A., MacNeil, I. A., Minor, C., Tiong, C. L., Gilman, M., Osburne, M. S., Clardy, J., Handelsman, J., Goodman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+the+Soil+Metagenomic:+A+Strategy+for+Accessing+the+Genetic+and+Functional+Diversity+of+Uncultured+Microorganisms.">Cloning the Soil Metagenomic: A Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms.</a> Appl. Environ. Microbiol. 66, 2541-2547 (2000).</li><li>Beja, O., Aravind, L., Koonin, E. V., Suzuki, M. T., Hadd, A., Nguyen, L. P., Jovanovich, S. B., Gates, C. M., Feldman, R. A., Spudich, J. L., Spudich, E. N., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Rhodopsin:+Evidence+for+a+New+Type+of+Phototrophy+in+the+Sea.">Bacterial Rhodopsin: Evidence for a New Type of Phototrophy in the Sea.</a> Science. 289, 1902-1906 (2000).</li></ol>

鉴于总细胞去除复杂和高度多样化的微生物垫样品是不实际的，首要关注的是如何提取的细胞代表的整体微生物垫界。在以前的研究中，微生物16S rRNA基因的PCR - DGGE技术分析显示，5个细胞去除在本议定书中所使用的步骤提取细胞，微生物席的整体社会7代表。细胞提取的实际步骤，需要提供一个整体的微生物群落的代表可能会改变取决于样品类型的细胞沉淀。对于不同样品的最佳协议发展...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> β-巯基乙醇</td><td> Sigma - Aldrich公司</td><td> M3148 </td><td></td></tr><tr><td>聚乙二醇8000 </td><td> Promega公司</td><td> V3011 </td><td> 20％在1.2 M氯化钠</td></tr><tr><td>醋酸钾</td><td> Fisher Scientific则</td><td></td><td> Fisher Scientific则</td></tr><tr><td> QUANT - dsDNA抗体检测套件</td><td> Invitrogen公司</td><td> Q33130 </td><td></td></tr><tr><td>核糖核酸酶</td><td>震中</td><td> MRNA092 </td><td></td></tr><tr><td>氯化钠</td><td> BDH化学品</td><td> BDH8014 </td><td>适当的浓度。 </td></tr><tr><td>十二烷基硫酸钠</td><td> Fisher Scientific则</td><td> 03-500-509 </td><td> 10％水</td></tr><tr><td>六偏磷酸钠</td><td>电解二氧化锰化学品</td><td> SX0583 - 3 </td><td>在水中的2％ </td></tr><tr><td> TBE </td><td> Fisher Scientific则</td><td> BP1333 - 1 </td><td></td></tr><tr><td>厨师映射XA系统</td><td> Bio - Rad公司实验室</td><td> 170-3670 </td><td></td></tr><tr><td> NanoDrop 1000分光光度计</td><td> Thermo Scientific的</td><td> ND - 1000 </td><td></td></tr><tr><td> Vortexer </td><td>科学工业公司</td><td></td><td></td></tr><tr><td>紫外线交联剂</td><td> UVP </td><td></td><td></td></tr><tr><td>华林搅拌器</td><td>华林实验室</td><td> LB10S </td><td></td></tr></tbody></table>

extraction of high molecular weight dna from microbial mats

宏基因组数据的成功和准确的分析和解释是依赖于高品质，高相对分子质量（HMW）社会DNA的高效提取。然而，环境垫样品往往造成困难，取得大量高品质，高分子量DNA的浓度。盐湖微生物席含有高含量的胞外聚合物（EPS）和盐，可能会抑制提取的DNA的下游应用。直接和严厉的方法经常被用来从难治性样本中的DNA提取。这些方法通常是因为EPS垫，胶粘剂基质，在直接裂解结合的DNA 2,3。严厉的提取方法，DNA为4,5,6小尺寸变得支离破碎。 的DNA，从而变得不适合大插入载体克隆。为了规避这些限制，我们报告一个改进的方法提取高分子量DNA质量好，数量从盐湖微生物席。我们采用一种间接的方法，涉及从微生物细胞通过混合和差速离心背景垫矩阵分离。使用的机械和化学程序的组合，从提取的微生物细胞的DNA提取和纯化。我们的协议，产量约2微克每克垫样品高分子量DNA（35-50 KB）， 与 A 260/280 1.6的比例。此外，扩增16S rRNA基因7表明，该协议是能够最大限度地减少或消除污染物的任何抑制作用。我们的研究结果提供了一个功能的宏基因组研究的微生物席的HMW DNA提取适当的方法和可能适用于其他环境样品，从DNA的提取是具有挑战性的的。

Watch this Scientific Journal Video about Extraction of High Molecular Weight DNA from Microbial Mats at JoVE.com

Extraction of High Molecular Weight DNA from Microbial Mats

我们提供了一个从盐湖微生物席超高分子量DNA提取的改进协议。微生物细胞中分离DNA提取和纯化前的垫矩阵。这将增强的浓度，质量和大小的DNA。该协议可用于其他耐火材料样品。

超高分子量DNA提取微生物席

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Research

JoVE Journal

Biology

Biology of Microbial Communities  - Interview

微生物群落的生物 - 面试

科研

生物学

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