1。ハイブリダイゼーションの手順ワンハイブリダイゼーションチューブは、1または2膜（22 cmの大きさ22）がかかります。ハイブリダイゼーション混合物を50mlとハイブリダイゼーションチューブあたりのDNAプローブ（10 ng / mlの最終濃度）の0.5mgを使用してください。最良の結果を得るには、プローブとしてゲル精製したDNAを使用してください。プローブ調製スケールアップした場合、チューブの数ではなく、チューブあたり試薬の量を増やすことを増やす。 プローブ調製（0.5mgのDNA）： <ol><li>水60μlで架橋剤溶液15μlを希釈する。 </li><li>スクリューキャップチューブに50μlの合計容積に水では10 ng /μlにDNAを希釈する。 </li><li>沸騰したお湯で5分間変性されたDNA。 </li><li>急速に氷に転送し、5分間冷ます。簡単にスピン。 </li><li>反応緩衝液50μlを加え、穏やかに混ぜる。 </li><li>標識試薬10μlを加え、穏やかに混ぜる。 </li><li>ステップ1から希釈架橋剤溶液50μlを加え、穏やかに混ぜる。穏やかに攪拌し、簡単にスピン。 </li><li> 37℃で30分間インキュベートします。 </li><li>最大2時間まで氷上で維持するか、-20℃で100％のグリセロールとストアの等量を追加する 注：キットに付属の水を使用してください。特に断りのない限り氷上のすべての試薬 ​​を保管してください。 </li></ol>膜調製とハイブリダイゼーション<ol><li>ハイブリダイゼーション緩衝液とハイブリダイゼーションチューブ60への予熱50ミリリットル℃に</li><li>ハイブリダイゼーションチューブにハイブリダイゼーションバッファーを追加します。膜（s）をロールアップするとハイブリダイゼーションチューブに加える。 （チューブあたり2膜、完全に一つの膜上最初のロールを追加する場合は、同じ方向に上に他の膜をロールアップします。） </li><li> 60℃のハイブリダイゼーションチューブをインキュベート℃で少なくとも半時間のためのCを（膜（s）が全身ずぶぬれになることを確認してください。） </li><li>ハイブリダイゼーションチューブから標識プローブを含むチューブに長いピペットでハイブリダイゼーション緩衝液約1mlを転送します。穏やかに攪拌し、簡単にスピン。ハイブリダイゼーションチューブに戻って混合物を移す。 </li><li> 60℃で一晩。 注：使用鉗子と手袋膜を処理する。あまりにも多くの攪拌は望ましくない泡の永続的な形成の原因となるので、ゆっくりとハイブリダイゼーションチューブのコンテンツを混ぜる。膜は湿潤状態で追加する必要があります。追加する前に、必要に応じて、ハイブリダイゼーション緩衝液または水に浸すと余分な水分を切る。 </li></ol> 2。検出の手順洗濯膜<ol><li> 60℃に予熱一次洗浄バッファー（電子レンジ及び/または加熱プレートを使用してください。） </li><li> oneハイブリダイゼーションチューブ2膜がある場合は、きれいに、予熱、空のハイブリダイゼーションチューブに一つの膜を転送する。 </li><li>簡単に言えば一次洗浄バッファーの約50 mlのハイブリダイゼーションチューブをリンス。 </li><li> 60ハイブリダイゼーションチューブとインキュベートする一次洗浄バッファーの約100 mlを加え℃で10分間。 </li><li>ステップ3と4を繰り返します。 </li><li> 5分間室温で二次洗浄バッファーの少なくとも250mlの容器とインキュベートし、洗浄するために膜を移し。 </li><li>手順6を繰り返します。 注：膜が信号生成の前に½時間まで室温で二次洗浄バッファーに保持されることがあります。 </li></ol>信号発生と検出（一つの膜のための手順） <ol><li>卓上へSaranWrapフラットのテープ2個（ステップ2の1つ、ステップ4のいずれか）。 </li><li>慎重に容器の側面に膜の角に触れることにより膜から余分なバッファを切るとSaranWrap（最大試料側）にメンブレンを転送する。 </li><li>膜上の膜と倍サランのラップ上ECF試薬6〜7 mlを注ぐ。慎重にゆっくりとSaranWrap以上キムワイプを移動することによって、細胞膜上のECFの試薬を販売しています。 5分間インキュベートする。 </li><li>メンブレンから過剰なECFの試薬を切ると新しいSaranWrap、膜上の倍のラップの上にそれを転送し、テープで縁をシール。アルミ箔をメンブレンにかぶせ。 1-2時間インキュベートする。 </li><li>ガラス板の上にECFの試薬の1〜2 mlを分散することにより、スキャナのガラス板を準備します。その後、ガラス板上に膜を転送する（下にサンプル側）と膜上のECFの試薬の別の1〜2ミリリットルを配布する。 SaranWrapとオーバーレイと慎重に離れて膜からすべての気泡を押してください。 （これは、ガラス板に押し付け膜を維持するためにSaranWrapの上に硬い板を置くことができます。） </li><li>検出フィルタLPB（510 nm）を、分解能は50μm、16ビットの信号を卒業、CH1 400 V.（スキャン、励起473 nmの：次の設定で富士FLA - 5100蛍光スキャナー（&quot;1レーザ1のイメージ&quot;モード）を使用してください22センチメートルでサイズ22の膜あたり約15〜20分かかります。） 省略可能： </li><li> 2〜4時間（以上）放置します。 </li><li>手順6のようにスキャンする手順を繰り返します。 </li></ol> 3。手順（NUF版）をストリッピングして保存<ol><li> 100％に膜を移しら穏やかに振盪しながら10分間室温でhanolとイ​​ンキュベート。 </li><li>新鮮な100％エタノールで置き換えると、室温で一晩インキュベートする。 （メンブレンは数日間エタノールに残っていることがあります。） </li><li>蒸留水でエタノールを交換し、10分間室温でインキュベートする。 </li><li>ハイブリダイゼーションチューブを100 mlの水と10％SDS（最後の0.5％SDS）を5mlを加え、穏やかに混ぜる。メンブレンを追加。 </li><li> 80℃1時間インキュベート</li><li>きれいな、空の容器にメンブレンを移し。 </li><li> SDS（​​最後の0.1％SDS）電子レンジでの活発な沸騰にビーカーと熱水500mlを加え、10％の5 mlです。 </li><li>膜上にほぼ沸騰0.1％SDS溶液を注ぎ、軽く数分間振とうする。室温に冷却してください。 </li><li>少なくとも10分間滅菌100mMトリスpHを8の少なくとも250 mlでリンス。 </li><li> SaranWrapの膜とテープで慎重に近いエッジを包みます。 4℃で保存します。 （膜が完全に乾くことはない必要があります。） </li></ol>

ストリッピングの手順が最適化されていません。しかし、メーカーの指示（2回洗浄それぞれ、100％エタノールで10分間、60℃0.5％SDSで1時間の1洗浄）で与えられるストリッピングの手順は不十分です。 100％エタノールで少なくとも一晩のインキュベーションでは、ECFの反応生成物を溶解するために必要であり、100％エタノールで数日のインキュベーションでは、メンブレンをリプローブする能力に悪影響が?...

組み合わせパックRPN3692 = RPN3680 + RPN3685

large-scale screens of metagenomic libraries

前の栽培を必要とせずに微生物から連続したゲノム配列のメタゲノムライブラリのアーカイブ大きい断片。メタゲノムライブラリーを作成し、スクリーニングするための合理化された手順を生成すると、未培養微生物群集の遺伝的および代謝特性に効率的な高スループットの調査に有用である。ここでは、主要なプロトコルは、我々は正確に交互にロボットの計測と（人間の）手動の介入に依存する長いプロセスを記述するための最も有用なフォーマットである提案するビデオ、上に表示されています。最初に、我々は、24 384ライブラリのプレートから重複したコロニーを含むことができるそれぞれの高密度マクロアレイメンブレン上にライブラリークローンを、見つけるためにロボットを採用。オートメーションは、高密度の空間的配置にライブラリークローンの大規模な数に合わせて必要な規模の小型化のためだけでなく、精度と速度のため、この手順が不可欠ですが、。一度生成された、我々は次のマクロアレイメンブレンは、プローブのラベリング、メンブレンハイブリダイゼーション、およびシグナル検出のための標準プロトコルの修正バージョンを使用して、目的の遺伝子をスクリーニングすることができる方法を示しました。我々は、ビデオと一緒にオンラインで利用可能なすべてが詳細に書かれた必要な手順の説明と必要な材料、これらの手順の視覚的なデモンストレーションを補完。

Watch this Scientific Journal Video about Large-Scale Screens of Metagenomic Libraries at JoVE.com

Large-Scale Screens of Metagenomic Libraries

メタゲノムライブラリの大規模スクリーン

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

large-scale-screens-of-metagenomic-libraries

Research

JoVE Journal

Biology

8.5K ビュー.  MIT - Massachusetts Institute of Technology. 

ビデオ: Large-Scale Screens of Metagenomic Libraries

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

全モンキー脳の大規模なタンパク質の検出のためのバッチの免疫染色

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

分離と成人ヒト内皮コロニー形成前駆細胞の大規模拡張

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

大規模ゼブラフィッシュベース in vivoで小分子スクリーン

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

メタゲノムライブラリからセルラーゼ活性をBiominingためのハイスループットスクリーニング

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

膵島質量、サイズ分布とアーキテクチャのコンピュータ支援大規模可視化と定量化

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

バーコード酵母ライブラリーの競争力のあるゲノム画面

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

大規模なSNPジェノタイピングアプリケーション用インフィニウムアッセイ

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

大規模な遺伝子ノックダウン中<em&gt; C.虫</em&gt;堅牢な機能喪失表現型を生成するためのdsRNA摂食ライブラリの使用

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感な、大規模定量的メタボロームのための戦略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY：酵母増殖の多パラメータ定量化のための大規模法

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...