1. Гибридизация процедуры Один гибридизации трубка будет принимать по 1 или 2 мембраны (размер 22 на 22 см). Используйте 50 мл гибридизации смеси и 0,5 мг ДНК-зонда (10 нг / мл, конечная концентрация) в гибридизации трубки. Для достижения наилучших результатов, используйте гель-очищенной ДНК в качестве зонда. Если расширение масштабов подготовки зонда, увеличение количества трубок, а не увеличения количества реагентов в трубке. Зонд подготовки (0,5 мг ДНК): <ol><li> Развести 15 мкл поперечных связей решение с 60 мкл воды. </li><li> Развести ДНК до 10 нг / мкл водой до 50 мкл общего объема в винт крышками микропробирок. </li><li> Денатурации ДНК в течение 5 минут в кипящую воду. </li><li> Быстро передавайте в лед и дайте остыть в течение 5 мин. Спиновые кратко. </li><li> Добавить 50 мкл реакционного буфера и аккуратно перемешать. </li><li> Добавьте 10 мкл реагента маркировки и аккуратно перемешать. </li><li> Добавить 50 мкл разбавленного поперечных связей решение с шага 1 и аккуратно перемешать. Vortex мягко и спина кратко. </li><li> Инкубировать при 37 ° С в течение 30 мин. </li><li> Держите на льду в течение до 2 ч или добавить равный объем 100% глицерина и хранить при температуре -20 ° C. Примечание: Использование воды, поставляемой в комплекте. Храните все реагенты на льду, если не указано иное. </li></ol> Мембранные подготовки и гибридизации <ol><li> Разогрейте 50 мл буфера гибридизации и гибридизации трубки до 60 ° C. </li><li> Добавить гибридизации буфера для гибридизации трубки. Свернуть мембраны (ов) и добавить к гибридизации трубки. (Если добавить 2 мембраны в трубку, сначала свернуть одной мембраны полностью, затем сверните другие мембраны на первое место в том же направлении.) </li><li> Инкубируйте гибридизации трубке при 60 ° С не менее ½ ч. (Удостоверьтесь, что мембрана (ы) промокнуть основательно.) </li><li> Передача около 1 мл буфера гибридизации с длинной пипеткой от гибридизации трубки в пробирку меченым зондом. Vortex мягко и спина кратко. Передача смесь обратно в гибридизации трубки. </li><li> Инкубировать при 60 ° С в течение ночи. Примечание: Используйте пинцет и перчатки при работе с оболочкой. Смешайте содержание гибридизации трубы аккуратно, потому что слишком много агитация вызовет постоянного образования нежелательных пены. Мембраны должны быть добавлены в мокром состоянии, замочить в буфер гибридизации или воды в случае необходимости и стечь лишней жидкости перед добавлением. </li></ol> 2. Определение процедуры Стиральная мембран <ol><li> Разогреть первичного буфера стиральную до 60 ° C. (Используйте микроволновую печь и / или нагревательного элемента.) </li><li> Если Есть две мембраны в одном гибридизации трубки, передача одной мембраны на чистую, разогретую, пустые трубки гибридизации. </li><li> Кратко промыть гибридизации трубки около 50 мл первичной промывочным буфером. </li><li> Добавить 100 мл первичной промывочный буфер в трубку гибридизации и инкубировать при температуре 60 ° С в течение 10 мин. </li><li> Повторите шаг 3 и 4. </li><li> Передача мембраны для мытья тары и инкубировать, по крайней мере 250 мл вторичный буфер стирка при комнатной температуре в течение 5 мин. </li><li> Повторите шаг 6. Примечание: Мембраны могут быть оставлены на вторичном буфере стирка при комнатной температуре в течение до ½ часа до генерации сигнала. </li></ol> Поколения и обнаружения сигнала (процедура для одной мембраны) <ol><li> Лента двух кусков плоских SaranWrap на настольные (по одному на шаге 2 и по одному на шаге 4). </li><li> Тщательно отвода избыточного буфер из мембраны, касаясь углов мембраны к стенке контейнера и передачи мембраны на SaranWrap (образец вверх). </li><li> Залить 6-7 мл ECF реагента над мембраной и раз Саран запахом мембраны. Тщательно распространять ECF реагента над мембраны, мягко движущихся Kimwipe более SaranWrap. Инкубировать в течение 5 мин. </li><li> Слить избыток реагента ECF от мембраны и передача его на новый SaranWrap, сложите запахом мембраны и уплотнения краев ленты. Обложка мембрана с алюминиевой фольгой. Инкубируйте в течение 1-2 часов. </li><li> Подготовка пластин стекла сканера, распределяя 1-2 мл ECF реагента над стеклянной пластиной. Затем перенесите мембраны на стеклянную пластину (образец стороной вниз) и распространять еще 1-2 мл реагента ECF над мембраной. Наложение с SaranWrap и осторожно подтолкнуть все пузырьки воздуха в сторону от мембраны. (Вы можете разместить жесткой пластины сверху SaranWrap держать мембрана прижимается к стеклянной пластинке.) </li><li> Используйте Fuji FLA-5100 флуоресценции сканер (&quot;1 лазер 1 изображение&quot; режим) со следующими параметрами: возбуждение 473 нм, обнаружение фильтр LPB (510 нм), разрешение 50 мкм, 16-битный сигнал окончания CH1 400 В. (сканирование занимает около 15-20 мин на оболочку размером 22 на 22 см). Дополнительно: </li><li> Дайте настояться в течение 2-4 часов (или дольше). </li><li> Повторите сканирование как в шаге 6. </li></ol> 3. Зачистка и хранения процедуры (Нуф версия) <ol><li> Передача мембраны до 100% и др.hanol и инкубировать при комнатной температуре в течение 10 мин при легком помешивании. </li><li> Замените свежие 100% этилового спирта и выдержать в течение ночи при комнатной температуре. (Мембрана может оставаться в этаноле в течение нескольких дней.) </li><li> Замените этанола с дистиллированной водой и инкубировать при комнатной температуре в течение 10 мин. </li><li> Добавить 100 мл воды и 5 мл 10% SDS (последние 0,5% SDS) для гибридизации трубки и аккуратно перемешать. Добавить мембраны. </li><li> Инкубировать при 80 ° С в течение 1 ч. </li><li> Передача мембраны чистый, пустой контейнер. </li><li> Добавить 500 мл воды и 5 мл 10% SDS (окончательный 0,1% SDS) стакан и тепло энергичный варить в микроволновой печи. </li><li> Налейте кипящую почти 0,1% SDS решением над мембраной и мягко агитировать за несколько минут. Дать остыть до комнатной температуры. </li><li> Полоскание в по меньшей мере 250 мл стерильного 100 мМ Трис рН 8, по крайней мере 10 мин. </li><li> Оберните мембраны в SaranWrap и тщательно близко краям ленты. Хранить при температуре 4 ° С. (Мембраны никогда не должна высыхать.) </li></ol>

Зачистки процедура не была оптимизирована. Тем не менее, зачистки процедурой, приведенной в инструкции производителя (2 моет каждый, 10 мин в 100% этанол, 1 мыть 1 ч при 60 ° С в 0,5% SDS) является недостаточной. По крайней мере инкубации в течение ночи в 100% этанола необходимо растворить продукт ECF реакции; нес...

Комбинация пакет RPN3692 = RPN3680 + RPN3685

large-scale screens of metagenomic libraries

Метагеномных архива библиотеки крупных фрагментов смежных геномных последовательностей из микроорганизмов, не требуя предварительного культивирования. Создание упрощенной процедуре для создания и проверки метагеномных библиотек поэтому полезен для эффективного высокопроизводительного расследования генетических и метаболических свойств некультурных комплексов микроорганизмов. Здесь основные протоколы представлены на видео, которое мы предлагаем, является наиболее полезным форматом для точного описания длительный процесс, который попеременно зависит от роботов приборов и (человеческого) вмешательства руководства. Во-первых, мы использовали робототехники обнаружить библиотеку клонов на высокой плотности мембран macroarray, каждый из которых может содержать повторяющиеся колоний от двадцати четырех 384-луночных библиотеки. Автоматизация имеет важное значение для этой процедуры не только точность и скорость, но и за счет миниатюризации масштабах, необходимых, чтобы соответствовать большое количество клонов в библиотеке высокой плотности пространственных механизмов. После генерируется, мы в следующий раз продемонстрировал, как macroarray мембраны могут пройти обследование на гены интереса с использованием модифицированной версии стандартных протоколов для зонда маркировки, мембранные гибридизация и обнаружения сигнала. Мы дополняли наглядной демонстрации этих процедур с подробного письменного описания этапов и материалов, необходимых, все из которых доступны в Интернете вместе с видео.

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

Large-Scale Screens of Metagenomic Libraries

Крупномасштабная Экраны метагеномных библиотеки

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

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JoVE Journal

Biology

8.5K Views.  MIT - Massachusetts Institute of Technology. 

Video: 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.

Пакетный Иммуноокрашивание крупномасштабных белка Обнаружение в весь мозг обезьяны

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

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.

Выделение и расширение добычи взрослых человеческих эндотелиальных клеток, образующих колонии Прародитель

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.

Крупномасштабные данио рерио основе В естественных условиях Малый экран Молекула

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.

Экран с высоким Пропускная Biomining Целлюлаза активность метагеномных из библиотеки

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.

Компьютерный Крупномасштабные визуализации и количественной оценки островков поджелудочной железы Масса, распределении размеров и архитектуры

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.

Конкурентные Геномная Экраны Barcoded Библиотеки дрожжей

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.

Infinium Анализ на крупномасштабных SNP генотипирования приложений

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.

Крупномасштабная Нокдаун гена в<em&gt; С. Элеганс</em&gt; Использование дцРНК кормления библиотек для генерации Прочные с потерей функции фенотипов

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.

Стратегия для чувствительной, крупномасштабного Количественные Metabolomics

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: крупномасштабный метод многопараметрического количественного определения роста дрожжей

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