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（涡精灵R 2的）（150转）从基质中的微生物细胞。 </li><li>在低速离心机（500X克）在4 ° C 15分钟轻轻地转移到一个烧瓶中，以最小的干扰沉积物上清。 </li><li>使用颗粒泥沙，重复融合和步骤，此外，在步骤1.3结束每个泥沙沉淀新鲜氯化钠1.2和1.3四个额外的时间。从每个氯化钠提取上清液转移到一个新瓶中。 </li><li> 4节5号电池在200毫升分装在15分钟的高速（25000 XG）的提取和离心机结合上清° C。弃上清，重悬细胞沉淀在每10毫升2％的六偏磷酸钠，结合颗粒，并用2％的六偏磷酸钠容至200 ml。晃动在室温为30分钟（150转）洗细胞。在此步骤结束时，你应该只有一个细胞管进行下一步。 </li><li>在4 ° C 15分钟高速离心（25000 XG）弃去上清液，重悬细胞沉淀的TE（50 mM的EDTA）200毫升，洗细胞晃动在室温为10分钟（150转）。 </li><li>在高速离心15分钟，4（25000 XG）℃，弃上清，悬浮在15毫升TE（10毫米EDTA），并存储200μL分装在-80℃直到进行DNA提取所需要的。 </li></ol> 2。 DNA提取和纯化： <ol><li>添加200μL，5 M氯化钠，200μL10％SDS，100μL，14.3中号β-巯基乙醇200μL微生物细胞等份。轻轻混匀反相4-6倍。 </li><li>主题的混合物3轮冻融液氮浸2分钟后在65 ° C为5分钟的水浴解冻。延长最终解冻10分钟。 </li><li>加入200μL的5个M醋酸钾（pH值5.5）和10分钟的冰地点。 XG离心机在10,000 10分钟，4℃，将上清转移到一个新的管使用大口径的枪头。添加3μL核糖核酸酶A和孵育37 ° C为1小时。 </li><li>加入等体积的氯仿，摇匀简单颠倒，在15000 XG离心机在室温下10分钟。 </li><li>小心地转移到一个新的管使用大口径的枪头上清的顶层。避免吸取白色接口层位于顶层和底层之间。重复氯仿萃取过程。 </li><li>加入同等体积的冰鲜（在-20 ° C）的异丙醇上清，在冰上孵育30分钟沉淀DNA。 </li><li> （20800 XG）最大速度离心10分钟，4 ° C至颗粒的DNA。 </li><li> 1毫升的冷冻（-20℃）70％的乙醇弃掉上清，洗DNA沉淀。 </li><li>最大速度（20800 XG）10分钟，4 ° C离心，弃上清液，重复10分钟70％的乙醇清洗，空气干燥DNA沉淀。重复70％的乙醇洗。 </li><li>悬浮在25μLTE（10毫米EDTA）的DNA，温在65 ° C为5分钟，合并成一管，如果有多个管已使用，而且化妆体积500μLTE。 </li><li>加入500μl冰冷的（或在4 ° C）20％聚乙二醇（PEG）（1.2 M氯化钠编制）。轻轻地反转，在冰上孵育10分钟的最高速度为10分钟，离心4 ° C混合沉淀DNA。取出并弃去上清液，用1毫升冷冻70％的乙醇，空气干燥10分钟沉淀DNA，并在30-50μLTE或分子级的水悬浮。由气候变暖促进再悬浮的DNA在65 ° C时，5分钟和商店的DNA -80 ° C。 </li></ol> 3。 DNA的纯度，浓度和大小测定： <ol><li>确定一个260/280和A 二百三分之二百六十零比使用Nanodrop 1000分光光度计或任何其他适当的仪器通过测量DNA的质量。 </li><li>确定DNA的浓度，根据制造商的指示使用QUANT - dsDNA抗体检测试剂盒。 </li><li>确定使用脉冲场凝胶电泳的DNA大小。准备用1％琼脂糖凝胶和使用0.5X TBE厨师映射的XA系统的下列参数; 0.35小号的初始开关时间，最后的切换时间的7.67小号，和120 °角为6.0 V / cm时梯度线性ř功放的因素。 30分钟的凝胶染色1 × SYBR GREEN我。负载之间700-1000 ng总DNA凝胶。见图。 3简明的形式为一个完整的协议。 </li></ol>代表性的成果： 细胞提取： 顺序微生物细胞提取物（上清）表明，提取物的浊度，提取增加的数量减少。这表明，在下面的每一个连续的细胞提取的细胞数量的减少。这里要注意的是，每个额外的细胞提取步骤污染物引进提供了机会，细胞提取的数量应尽量减少，最终使细胞沉淀是代表整体的微生物群落。其他来源的污染控制，通过采取适当的实验室无菌技术。例如，解决方案是准备在灭菌去离子水和过滤消毒。容器用酒精消毒，灭菌，紫外光照射下，紫外线交联剂治疗。 DNA的浓度和质量的决心： 一个260/280的比例为1.6，该协议产生高分子量DNA（35-50）（图4）约2微克每克垫样品和A 二百三十〇分之二百六十○0.7的比例（见表1） 。虽然出现了一个二百三十分之二百六比例要低，没有抑制下游分子为基础的应用程序，如在另一项研究中 7中观察到的16S rRNA基因的PCR扩增。重要的是要注意从盐湖垫DNA有两个主要的污染来源; EPS和盐。因此，这些污染物的痕迹，尽管作出了巨大努力，以减少对下游应用的影响可能会影响的一个二百三十分之二百六十零比率。 DNA大小的决心： 脉冲场凝胶电泳在DNA检查，如在图3所示的间歇性定向交换机采用了脉动电流流。我们的协议产生了一个约35-50 kb的高分子量的DNA。虽然一些DNA的大小可能会小于30 KB，重要的是要涂抹上述35 KB fosmid克隆〜40 kb的DNA插入和较大的DNA片段，提供了更大的获得完整的生物合成途径的一部分。在我们的研究中，上述35 KB的DNA涂抹被切除大型插入载体克隆和其他分子应用纯化。 <img src="/files/ftp_upload/2887/2887fig1.jpg" alt="图1"/> 图1。盐湖微生物垫采样点（大池）位于伊柳塞拉，巴哈马。 <img src="/files/ftp_upload/2887/2887fig2.jpg" alt="图2"/> 图2：横截面在这项研究中所使用的高盐度的微生物垫。从伊柳塞拉，巴哈马位于盐湖池塘微生物垫。 <img src="/files/ftp_upload/2887/2887fig3.jpg" alt="图3"/> （三）涉及微生物细胞提取细胞裂解液，提取，和宏基因组DNA的纯化过程示意图<a href="http://www.jove.com/files/ftp_upload/2887/2887fig3large.jpg">代表性。点击这里查看一个较大的</a>图像。 <img src="/files/ftp_upload/2887/2887fig4.jpg" alt="图4"/> 图4。使用脉冲场凝胶电泳对提取的DNA分子量表征。巷M是一种高分子量的标记，泳道1和2的宏基因组DNA的提取使用上述协议的伊柳塞拉盐湖垫复制。 <table border="1"><tbody><tr><td> 提取方法 </td><td></td><td> 浓度纳克/克 </td><td> 一个260/280 </td><td> 一个二百三分之二百六十〇 </td></tr><tr><td> 聚乙二醇 </td><td>精华1 </td><td> 1806.1 </td><td> 1.64 </td><td> 0.76 </td></tr><tr><td></td><td>精华2 </td><td> 2010.8 </td><td> 1.61 </td><td> 0.75 </td></tr></tbody></table> 表1。提取的DNA的浓度和质量的测量微生物盐湖垫。

<ol>
	<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 ...

extraction-of-high-molecular-weight-dna-from-microbial-mats

Research

JoVE Journal

Biology

19.4K 次观看.  Arnold School of Public Health, University of South Carolina. 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。

视频: Extraction of High Molecular Weight DNA from Microbial Mats

Biology of Microbial Communities  - Interview

微生物群落的生物 - 面试

科研

生物学

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

DNA Extraction from 0.22 &amp;mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

从0.22微米Sterivex过滤器和氯化铯密度梯度离心法的DNA提取

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

超高分子量基因组DNA的提取土壤和沉积物

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

粘蛋白琼脂糖凝胶电泳：蛋白质印迹高分子量糖蛋白


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

福尔马林固定石蜡包埋的组织核心的制备RNA和DNA提取

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

改进的高通量DNA提取板加载方法，降低污染的可能性

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

小规模提取 秀丽隐杆线虫 基因组DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...