This study was supported as basic research by CREST from the Japan Science and Technology Agency (JST) and by grants from JSPS/MEXT KAKENHI (Number 26291083) and the Canon Foundation to M.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

注:此协议不涉及水样采集和metabarcoding方法。水可以以不同的方式取决于研究的目的16进行采样，看看宫等 12使用MiFish引物metabarcoding方法的细节。注意，采样水应保持非常冷，并在数小时内过滤，以避免的eDNA的降解。另请注意，该协议涉及使用旋转振荡器和培养箱的，而后者必须足够大，以容纳前者。此外，离心分离机，可容纳均为15和50ml锥形管是不可缺少的，从过滤后的过滤器除去残留的液体和分别暗盒内以收集提取的DNA。 1.处理螺帽和1升的塑料袋注:如果过滤量&gt; 4 L.跳过此步骤<ol><li>通过一个螺丝c的中心钻一个孔AP（附连到一次性1升塑料袋）具有相同的直径（4.8 MM）作为管从阳路厄锁连接器突出。使用斜口钳，缩短阳路厄锁连接器的管以适当的长度（ 约 3毫米），以避免水的过滤后的帽内有少量的堵塞。 </li><li>申请专门用于聚乙烯（PE）和聚丙烯（PP）的粘合剂胶水的阳路厄锁连接器的底部和表面都和螺旋盖，分别。等待好的粘接几分钟（参照制造商的说明）。 </li><li>插入阳鲁尔锁连接器插入螺丝帽的孔。等到两部分（通常&gt; 24小时）的完整的粘接。用消毒使用前用10％的市售漂白剂（ 约 0.6％次氯酸钠）阳路厄锁连接器上的螺丝帽。 </li><li>冲在1升塑料袋的两个底部角落的两个孔，以从一个MES挂ħ面板（步骤3）。确保该孔的直径比所述网格板上的齿尖的尺寸。 </li></ol> 2.过滤系统的装配<ol><li>附加在高真空油管抽吸泵的输入连接器和管的另一端连接到一个歧管。可以肯定的歧管的三个红色叔阀处于"关闭位置"（ 即 ，横向）。 </li><li>戴着干净组手套，插入一个注射器阴配件，并在真空橡胶管用于过滤的顶端和底端的真空连接器，分别。 </li><li>仔细附加注射器阴配件的滤筒的出口和真空连接器连接到一个硅胶塞。 </li></ol> 3.使用滤芯水样（≤4L）的过滤注:如果过滤量&gt; 4 L.该过滤系统跳过此步骤需要自立F组或悬挂塑料袋装满1升的水。网状面板，多叉，而对于面板的立场，都可以从网上商店，将是组装这个单位非常有用。高压釜的入口和出口的路厄氏帽在使用前过滤筒。 <ol><li>倒入1升采样水入塑料袋中并与阳鲁尔锁连接器关闭螺旋盖。 </li><li>小心地用塑料袋的阳路厄锁定连接器连接的组装过滤器滤芯的流入口。不要过度拧紧鲁尔锁连接器;否则一旦开始抽水设备将泄漏。确保所有的连接都从网状面板挂塑料袋之前的安全。 </li><li>小心地用滤筒从两个叉悬挂网格面板上的塑料袋中，并插入一个硅胶塞进入歧管的入口。 </li><li>打开抽吸器。打开红色T阀过滤。运行歧管，直到滤芯干燥，T母鸡将红色叔阀到关闭位置。 </li><li>重复3.1-3.4步骤，直到水的所需量的过滤。 注:为每升从亚热带珊瑚礁取水，它需要的过滤约三分钟。 </li><li>小心地从塑料袋和真空连接器的滤芯。 </li><li>帽与入口和出口的路厄氏帽上的卡盒的两端。 </li><li>标签盒和入口路厄帽适当地使用溶剂型笔快干和非涂抹标记。 </li><li> DNA提取之前储存在-20℃的滤筒。 </li></ol> 4.水过滤（&gt; 4 L）采用滤芯样品注意:跳过这一步，如果水过滤体积是≤4L。该过滤系统需要配备一个阀门和一次性10毫升移液管尖端的10升书瓶中。的10毫升吸管尖端（15.0毫米）内径和尖端的锥形端应该适合的阀（15.0毫米）的外直径和过滤器滤芯的流入口，分别。这两个连接在一摩擦配合在过滤过程中牢固地保持。用消毒使用前10％市售漂白剂（ 约 0.6％次氯酸钠）枪头。 <ol><li>在书瓶准备的采样适量水。紧紧插入10毫升吸管尖端放入10升本书瓶的阀门。 </li><li>紧紧插入滤筒插入吸移管前端的出口并插入硅胶塞进入歧管的进气口。打开抽吸器。打开红色T阀过滤。运行所述歧管，直到滤筒是干的，然后打开红色叔阀到关闭位置。 注:对于10升从亚热带外珊瑚礁取海水，它需要的过滤约30-40分钟。 </li><li>小心地从塑料袋和真空连接器的滤芯。这两个帽与入口和出口的路厄氏帽上的卡盒的结束。标签盒和入口路厄帽适当地使用溶剂型笔快干和非涂抹标记。在DNA提取之前-20℃保存滤芯。 </li></ol>从过滤器埃德娜5.提取注:在步骤4和5，我们使用商业套件，主要针对以下"核血"的套件中提供的协议。为简单起见，我们将描述用于处理单个盒的过程。在实践中，我们建议在一个时间处理8（或离心机的最大数目）或更少的过滤器。 <ol><li>预热的孵化器56℃。 </li><li>通过从管切割铰链式盖制备2.0毫升管。丢弃上限。 注:此管被用作用于在过滤器中剩余的液体的收集管。 </li><li>从过滤器滤芯的入口（未出口）鲁尔盖，将进气口我n要收集管。紧密地密封采用自密封膜盒和收集管之间的连接。 </li><li>将合并的单元成15毫升锥形管离心适配器。离心盒在5000 xg离心1分钟，以去除剩余的液体在过滤器。 </li><li>从滤筒移除收集管并弃去管。重新盖盒的入口。 </li><li>制备的20微升蛋白酶K溶液的混合物中，220微升PBS（磷酸盐缓冲盐水;不是由试剂盒提供）和200微升缓冲液AL。 注:滤芯可容纳该混合物的四倍体积（ 加利福尼亚州 1.8ml）中。 </li><li>卸下入口盖和该混合物（440微升）加入到用枪头滤筒。插入吸移管完全插入进气口，使得枪头是盒刚好在膜上方内可见。 </li><li>重新盖上的进气口，然后将过滤器的车盒在旋转摇床上。 </li><li>放置在预热的培养箱的旋转振荡器在56℃和20 rpm的速度打开摇动20分钟。 </li><li>在温育后，准备一个新的2.0毫升管，标号管子适当地使用溶剂型笔，并将其放置在50ml的锥形管中。 注:前者（2.0毫升）和后者管（50毫升）被用于提取的DNA的收集和用于保持2.0毫升管，分别。 </li><li>培养后，除去入口盖和盒的端口插入入口（未出口）进50毫升的锥形管内2.0毫升管。用螺丝帽关闭50ml锥形管中。 </li><li>离心50毫升锥形管中，在5000 xg离心1分钟，以收集从盒中提取的DNA。移除滤筒并使用无菌镊子2.0毫升管和帽的管中。丢弃滤筒。 </li></ol> 6.纯化提取的DNA 注意:我们洗脱的eDNA用100微升缓冲AE，而不是商业套件手册中规定的200微升。 <ol><li>加入200μl乙醇（96-100％），以所提取的DNA在2.0毫升试管从上述步骤（ 约 440微升），并通过涡旋调匀。 </li><li>吸管将混合物成放置在2 ml收集管旋转柱。离心在5000 xg离心1分钟。丢弃流通和收集管。 </li><li>放置旋转柱在新的2ml收集管中，在5000×g下加入500μl缓冲液AW1，离心1分钟。丢弃流通和收集管。 </li><li>放置旋转柱在新的2ml收集管中，在20,000×加入500微升缓冲液AW2，与离心3分钟g。丢弃流通和收集管。 </li><li>准备一个新的1.5毫升管（未由试剂盒提供），并适当地使用快干和不涂抹标记防水笔标记管。转移自旋T列Ø1.5 ml管。 </li><li>通过加入100μl的缓冲液AE到离心柱膜的中央洗脱DNA。孵育在室温下1分钟，然后在6000 xg离心离心1分钟。 </li><li>丢弃旋转柱和帽的管中。储存在-20℃的纯化的DNA。 </li></ol>

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Genet. 17, 1-17 (2016).</li><li>Sorokulova, I., Olsen, E., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopolymers+for+sample+collection,+protection,+and+preservation.">Biopolymers for sample collection, protection, and preservation.</a> Appl. Microbiol. Biotechnol. 99, 5397-5406 (2015).</li><li>Renshaw, M. A., Olds, B. P., Jerde, C. L., McVeigh, M. M., Lodge, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+room+temperature+preservation+of+filtered+environmental+DNA+samples+and+assimilation+into+a+Phenol-Chloroform-Isoamyl+alcohol+DNA+extraction.">The room temperature preservation of filtered environmental DNA samples and assimilation into a Phenol-Chloroform-Isoamyl alcohol DNA extraction.</a> Mol. Ecol. Res. 2014, (2014).</li></ol>

The authors have nothing to disclose.

在使用环境样品，例如水和土壤许多metabarcoding研究中，过滤器滤芯的过滤后处理通常如下24,25:1）切开或开裂用手动工具（管切割器或钳子）的外壳; 2）清除墨盒上的过滤器;和3）切割所述滤波器小块用刀片用于DNA提取。为了避免过滤器滤芯的外壳内的这种麻烦的和费时的，它们很容易在实验室污染的程序，我们尝试几种DNA提取方法使用一种广泛使用的，价格低廉市售的试剂盒，并成功地?...

在水生环境环境DNA（的eDNA）是指水体中发现的遗传物质。最近的研究表明的eDNA的效用从各种水生环境，包括池塘1-3，4-8江河鱼类检测，溪流9，和海水10-14。这些研究大多集中在检测的单个或几个侵入1,4-6,8,14和珍稀或濒危物种3,9，而最近的一些研究试图在当地鱼类群落7,9多个物种的同时检测， 12,13,15和生物群落11,12。 后一种方法被称为"metabarcoding"和的eDNA metabarcoding使用的PCR引物的一个或多个集合横跨分类学上不同的样品coamplify的基因区域。其次是与索引和适配器此外文库制备和索引库由一个高通量平行测序分析平台。最近宫等人 12开发的通用PCR引物用于从鱼类（称为"MiFish"）metabarcoding的eDNA。所述MiFish引物靶向线粒体12S rRNA基因（163-185碱基），它包含足够的信息，以确定鱼生物分类科，属和除了一些密切相关的同源物种的高变区。随着使用中的eDNA metabarcoding的引物，美哉等 12检测到水族箱230余属亚热带海洋物种，水族馆附近的已知物种组成和珊瑚礁。 同时优化metabarcoding协议容纳天然海水以改变来自鱼类的eDNA浓度的水平，我们已经注意到，MiFish引物偶尔未能扩增后续文库制备目标区域。一个为这个不成功的PCR扩增更可能的原因是缺乏足够数量的TE的中所含的水的小体积的过滤mplate的DNA（ 即 1-2升）。虽然从特定分类群的eDNA浓度放大之前是不可知的大型水量（&gt; 1-2升），过滤是一个简单而有效的手段，从稀缺的鱼类数量和生物量，比如水环境收集更多的eDNA公海和深海生态系统。 相对于磁盘纤维过滤器在许多鱼的eDNA研究16的常规使用的，滤芯具有堵塞17之前容纳较大水量的优点。事实上，最近的一项研究表明，大体积（&gt; 20 L）采用滤芯18沿海海水样品的过滤。此外，它们被单独包装和无菌，实验工作流的若干步骤可以在过滤器外壳中进行，从而降低了从实验室19污染的可能性。后者特征为的eDNA metabarcoding关键，其中污染的风险仍然之间的最大实验挑战20,21。尽管滤芯这些技术优势，它尚未在鱼类的eDNA研究中使用的有两个例外8,15。 在这里，我们提供了从它的过滤器的eDNA的滤筒和提取水样的过滤的协议，而不必切开壳体。我们还提供了根据不同的水量（≤4L或&gt; 4升）两种可供选择的水过滤系统。为了比较新开发协议的性能，在我们的研究小组使用12,14,22,23玻璃纤维过滤先前使用的协议，我们执行的eDNA metabarcoding海水分析从一个巨大的鱼缸（7 500 立方米 ）与已知的物种组成，并示出了从两个协议作为代表性结果得出检测物种的数目。该协议ħ作为已经开发了用于从鱼类metabarcoding的eDNA，但也适用于的eDNA从其他生物体。

<table><tbody><tr><td>Mesh panel</td><td>Iris Ohyama</td><td>MPP-3060-BE</td><td></td></tr><tr><td>Metal prong</td><td>Iris Ohyama</td><td>MR12F</td><td></td></tr><tr><td>Stand for the mesh panel</td><td>No brand</td><td>4184-9507</td><td>available from Amazon Japan</td></tr><tr><td>1 L plastic bag with screw cap</td><td>Yanagi</td><td>DP16-TN1000</td><td></td></tr><tr><td>Male luer-lock connector</td><td>ISIS</td><td>11620</td><td></td></tr><tr><td>10 ml pipette tip</td><td>Eppendorf</td><td>0030 000.765</td><td></td></tr><tr><td>10 L book bottle with valve</td><td>As One</td><td>1-2169-01</td><td></td></tr><tr><td>Sterivex-HV filter</td><td>Millipore</td><td>SVHVL10RC</td><td>denoted as &quot;filter cartridge&quot; throughout the ms and used in the protocol</td></tr><tr><td>Male luer fitting</td><td>As One</td><td>1-7379-04</td><td></td></tr><tr><td>Female luer fitting</td><td>As One</td><td>5-1043-14&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Inlet luer cap</td><td>ISIS</td><td>VRMP6</td><td></td></tr><tr><td>Outlet luer cap</td><td>ISIS</td><td>VRFP6</td><td></td></tr><tr><td>High vacuum tubing</td><td>As One</td><td>6-590-01</td><td></td></tr><tr><td>Vacuum connector</td><td>As One</td><td>6-663-02</td><td></td></tr><tr><td>Silicone stopper</td><td>As One</td><td>1-7650-07</td><td></td></tr><tr><td>Manifold</td><td>As One</td><td>2-258-01</td><td></td></tr><tr><td>Aspirator-GAS-1</td><td>As One</td><td>1-7483-21</td><td></td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kit (250)</td><td>Qiagen</td><td>69506</td><td></td></tr><tr><td>PowerWater Sterivex DNA Isolation Kit</td><td>MO BIO</td><td>14600-50-NF</td><td>denoted as &quot;optional kit&quot; in the ms</td></tr><tr><td>Tabletop Centrifuge</td><td>Kubota</td><td>Model 4000</td><td>Maximum speed 6,000 rpm</td></tr><tr><td>Fixed-angle rotor</td><td>Kubota</td><td>AT-508C</td><td></td></tr><tr><td>Adaptor for a 15 ml conical tube</td><td>Kubota</td><td>055-1280</td><td></td></tr><tr><td>RNAlater Stabilization Solution</td><td>Thermo Fisher Scientific</td><td>AM7020</td><td></td></tr><tr><td></td><td>Parafilm</td><td>PM992</td><td>denoted as &quot;self-sealing film&quot;</td></tr></tbody></table>

use of a filter cartridge for filtration of water samples and extraction of environmental dna

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

这在技术上是难以分离和量化从所提取的散装的eDNA仅鱼的eDNA，因为MiFish引物从一些非鱼脊椎动物，如鸟类和哺乳动物coamplify目标区域，与同样大小的PCR产物（ 约 170 bp的）12。相反，量化鱼埃德娜，我们执行MiFish从使用过滤和DNA提取的两种不同的方法已知的物种组成的水族箱metabarcoding埃德娜分析，并从两个协议比较每个库检测物种的数量。这个简单的实验?...

Watch this Scientific Journal Video about Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA at JoVE.com

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

滤芯水过滤样品和环境DNA的提取使用

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

use-filter-cartridge-for-filtration-water-samples-extraction

Research

JoVE Journal

Environment

24.9K 次观看.  Natural History Museum and Institute, Chiba. We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

视频: Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

揭开看不见的玩家在海洋 - 一个领域指南水化学和海洋微生物

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

科研

环境科学

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

对水样的自动固相萃取小污染物的免疫学分析的简单方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

内毒素和DNA含量的空气采样滤波器分析

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species including invasive and (or) species at risk is the goal, typically accomplished by testing water and sediment for the presence of characteristic DNA signatures. Reliable and efficient procedures for the capture of eDNA are required, especially those that can be performed easily in the field by personnel with limited training and citizen scientists. The capture of eDNA using membrane filtration is widely used currently. This approach has inherent issues that include the choice of filter material and porosity, filter fouling, and time required on site for the process to be performed. Flocculation offers an alternative that can be easily implemented and applied to sampling regimes that strive to cover broad territories in limited time.

Capture of Environmental DNA eDNA from Water Samples by Flocculation

Environmental DNA (eDNA) is typically captured from water samples using alcohol precipitation, filtration, or flocculation. Presented here is an alternative protocol that uses lanthanum chloride to enable collection of dissolved and particulate-bound nucleic acids from water samples containing eukaryotic cells, prokaryotic cells, and virus.

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species ...

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil and gas. This process revolutionized the U.S. energy industry, as it gives access to resources that were previously unobtainable and now produces two-thirds of the total natural gas in the United States. Although fracking has had a positive impact on the U.S. economy, several studies have highlighted its detrimental environmental effects. Of particular concern is the effect of fracking on headwater streams, which are especially important due to their disproportionately large impact on the health of the entire watershed. The bacteria within those streams can be used as indicators of stream health, as the bacteria present and their abundance in a disturbed stream would be expected to differ from those in an otherwise comparable but undisturbed stream. Therefore, this protocol aims to use the bacterial community to determine if streams have been impacted by fracking. To this end, sediment, and water samples, from streams near fracking (potentially impacted) and upstream or in a different watershed of fracking activity (unimpacted) must be collected. Those samples are then subjected to nucleic acid extraction, library preparation, and sequencing to investigate microbial community composition. Correlational analysis and machine learning models can subsequently be employed to identify which features are explanative of variation in the community, as well as identification of predictive biomarkers for fracking&#39;s impact. These methods can reveal a variety of differences in the microbial communities among headwater streams, based on the proximity to fracking, and serve as a foundation for future investigations on the environmental impact of fracking activities.

Here, we present a protocol to investigate the impacts of hydraulic fracturing on nearby streams by analyzing their water and sediment microbial communities.

使用微生物分子特征评估水力压裂对溪流的影响

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil ...

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.

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 ...

生物学

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 ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

使用改良试剂盒进行经济的硅藻 DNA 提取

Extraction of Diatom DNA from Water Samples and Tissues

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

作者聚焦： 用于法医溺水检查的硅藻检测 

This article describes a protocol for diatom DNA extraction using a modified common DNA extraction kit.

从水样和组织中提取硅藻DNA

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...