1. 하이브 리다이 제이션 절차 하나의 하이브리드화 관은 1 또는 2 세포막을 (22cm로 크기가 22) 소요됩니다. 하이브 리다이 제이션 혼합물의 50 ML 및 하이브리드화 관 당 DNA 프로브 (10 NG / ML 최종 농도) 0.5 MG를 사용하십시오. 최상의 결과를 얻으려면, 프로브로 젤 - 정화 DNA를 사용합니다. 프로브 준비 최대 배율 경우, 튜브의 개수보다는 튜브 당 시약의 양을 증가를 향상시킬 수 있습니다. 프로브 준비 (0.5 MG DNA) : <ol><li> 물 60 μl와 가교제 용액 15 μl를 희석. </li><li> DNA 10 NG / 나사 덮인 microtube에 50 μl 전체 볼륨에 물을 μl를 희석. </li><li> 끓는 물에 5 분 변성의 DNA. </li><li> 신속하게 얼음을 전송하고 5 분 식지. 간단히 스핀. </li><li> 반응 완충액 50 μl를 추가하고 부드럽게 섞는다. </li><li> 시약 라벨 10 μl를 추가하고 부드럽게 섞는다. </li><li> 1 단계의 희석 가교제 용액 50 μl를 추가하고 부드럽게 섞는다. 부드럽게 소용돌이와 짧게 스핀. </li><li> 37 ° C 30 분 알을 품다. </li><li> 최대 2 H 위해 얼음에 보관 또는 -20 ° C. 100 %의 글리세롤과 저장소의 동일한 음량을 추가 참고 : 키트와 함께 제공된 물을 사용하십시오. 달리 명시되지 않는 한 얼음의 모든 시약 보관하십시오. </li></ol> 막 준비와 하이브리드화 <ol><li> 60 하이브 리다이 제이션 버퍼와 하이브리드화 관 앞서 열을 가하다 50 ML은 ° C. </li><li> 하이브리드화 튜브에 하이브리드화 버퍼를 추가합니다. 멤브레인 (S)을 올리고 하이브리드화 튜브에 추가합니다. (튜브 당 두 세포막, 완벽 한 멤브레인 첫번째 롤을 추가한다면, 같은 방향으로 상단에있는 다른 멤브레인을 롤.) </li><li> 60 하이브 리다이 제이션 튜브를 품어 ° 이상에 대한 C ½ H. (멤브레인 (S)가 완전히 젖었을 확인하십시오.) </li><li> 하이브 리다이 제이션 관에서 라벨 프로브를 포함하는 튜브에 긴 피펫과 하이브리드화 버퍼의 약 1 ML을 전송합니다. 부드럽게 소용돌이와 짧게 스핀. 하이브 리다이 제이션 튜브로 혼합물을 전송합니다. </li><li> ° C 하루 60 알을 품다. 참고 : 사용 포셉 및 장갑 세포막을 취급. 하이브리드화 튜브의 혼합 내용을 부드럽게하기 때문에 너무 많은 동요가 원하지 않는 거품의 영구적인 형성을 초래하게됩니다. 세포막은 서부 유럽 표준시 조건에 추가되어야, 하이브 리다이 제이션 버퍼 또는 물 필요한 경우에 담그다 및 추가하기 전에 여분의 액체를 배수. </li></ol> 2. 검색 절차 워싱 세포막 <ol><li> 60 ° C.에 앞서 열을 가하다 주 세척 버퍼 (전자렌지 및 / 또는 열판을 사용합니다.) </li><li> 한 하이브리드화 튜브 2 점막이있다면, 깨끗하고, preheated, 빈 하이브리드화 관 한 멤브레인을 전송하기만하면됩니다. </li><li> 간단히 차 세척 버퍼의 약 50 ML과 하이브리드화 튜브를 헹굴. </li><li> 60 하이브리드화 튜브와 부화 ° 10 분 C로 기본 세척 버퍼의 약 100 ML을 추가합니다. </li><li> 3, 4 단계를 반복합니다. </li><li> 5 분 실온에서 차 세척 버퍼 최소한 250 ML과 컨테이너와 부화를 세탁하기 위해 막을 전송. </li><li> 6 단계를 반복합니다. 참고 : 점막이 신호 생성하기 전에 ½ H로 최대 상온에서 보조 씻어 버퍼에 보관 수 있습니다. </li></ol> 신호 생성 및 검출 (한 막 절차) <ol><li> 탁상에 SaranWrap 평면의 테이프 두 조각 (2 단계에 대해 하나와 4 단계에 대해 하나). </li><li> 조심스럽게 용기의 측면에 대한 막의 모서리를 만져 멤브레인에서 여분의 버퍼를 분출과 SaranWrap (최대 샘플 사이드)에 멤브레인를 전송합니다. </li><li> 막 이상의 막, 접이식 비닐랩을 통해 ECF 시약의 6-7 ML 하거라. 조심스럽게 부드럽게 SaranWrap 통해 Kimwipe를 이동하여 막 이상 ECF 시약을 배포합니다. 5 분 알을 품다. </li><li> 멤브레인에서 초과 ECF 시약을 드레인과 새로운 SaranWrap, 멤브레인 위에 접어 버리고,에 그것을 전송하고 테이프로 가장자리를 밀봉. 알루미늄 호일로 멤브레인 표지. 1-2시간을위한 부화. </li><li> 유리 접시 이상의 ECF 시약 1-2 ML을 배포하여 스캐너 유리 플레이트를 준비합니다. 그런 다음 유리 플레이트 (아래 샘플 사이드)에 멤브레인를 전송하고 멤브레인 이상 ECF 시약의 또 다른 1-2 ML을 배포합니다. SaranWrap와 오버레이 조심스럽게 거리 멤브레인에서 공기 방울을 밀어. (당신은 유리 플레이트에 대한 누르면 막을 유지 SaranWrap 위에 성실히 접시를 배치할 수 있습니다.) </li><li> 여기 473 NM, 탐지 필터 LPB (510 NM), 해상도 50 μm의, 16 비트 신호를 졸업, CH1 400 V. (스캔 다음 설정으로 후지 FLA - 5100 형광 스캐너 ( &quot;1 레이저 한 이미지&quot;모드)를 사용 22cm의 크기는 22 멤브레인 당 약 15-20 분 정도 소요됩니다.) 옵션 : </li><li> 2~4시간 (이상)에 앉아 보자. </li><li> 6 단계 같이 스캔 반복합니다. </li></ol> 3. (NUF 버전) 스트립과 절차를 저장할 <ol><li> 백퍼센트 동부 표준시로 막을 전송부드러운 교반 10 분 실온에서 hanol와 부화. </li><li> 신선한 100 % 에탄올로 대체하고 실온에서 하룻밤 품어. (분리막은 며칠 동안 에탄올에 남아있을 수 있습니다.) </li><li> 증류수와 에탄올로 교체 10 분 실온에서 알을 품다. </li><li> SDS (최종 0.5 % SDS) 하이브리드화 튜브 100 ML 물 10 % 5 ML을 추가하고 부드럽게 섞는다. 멤브레인를 추가합니다. </li><li> 80 ° C 1 H.위한 부화 </li><li> 깨끗한 빈 컨테이너로 막은을 전송. </li><li> 10% SDS 500 ML 물 5 ML (최종 0.1 % SDS)을 전자렌지에 활발한 끓일 수있는 비커와 열을 추가합니다. </li><li> 멤브레인 위에 가까운 끓는 0.1 % SDS 용액을 붓고 부드럽게 몇 분 동안 선동. 실온으로 냉각하도록 허용합니다. </li><li> 적어도 10 분 멸균 100 MM 트리스 산도 8 적어도 250 ML에 씻어. </li><li> 랩 SaranWrap에 막과 테이프로 조심스럽게 닫습니다 가장자리. 4 스토어 ° C. (세포막 밖으로 건조 절대 필요합니다.) </li></ol>

스트립 절차는 최적화되지 않았습니다. 그러나, 제조 업체의 지침 (2 세척 각각 100 % 에탄올에 10 분, 60 ° C 0.5 %의 SDS 1 H 1 씻어)에 주어진 스트립 절차가 부족합니다. 100 % 에탄올에 적어도 하룻밤 배양은 ECF의 반응 생성물을 분해하는 데 필요한, ​​100 % 에탄올에 며칠 보육은 막을 reprobe 수있는 능력에 아무런 악영향이없는 것으로 나타납니다. 제조 업체의 SDS 치료도 부족 것으로 보인다. NUF 80 1 H를 시도했다 ° C 0...

large-scale screens of metagenomic libraries

이전 경작하지 않고 미생물의 게놈 시퀀스의 연속 Metagenomic 라이브러리 아카이브 큰 조각. metagenomic 라이브러리를 생성하고 검사를위한 간소화된 절차를 생성하는 것은 미개한 미생물 assemblages의 유전과 신진 대사 특성에 효율적으로 높은 처리량을 수사 따라서 유용합니다. 여기, 주요 프로토콜은 우리가 정확하게 교대 로봇 계측과 (인간) 수동 개입에 따라 긴 프로세스를 설명하기위한 가장 유용한 형식입니다 제안 비디오에 제공됩니다. 첫째, 우리는 스물네 384 자 도서관 접시에서 중복 식민지를 포함할 수 있습니다 각각의 고밀도 macroarray의 점막에 라이브러리 클론을 지점으로 로봇을 고용. 자동화는 또한 매우 치밀한 공간 준비로 도서관 클론의 다수에 맞게 필요한 규모의 소형화에 의해뿐만 아니라 정확성과 속도에 대해이 절차를 위해 필수적입니다,하지만. 일단 생성, 우리는 다음 macroarray의 점막이 프로브 라벨, 멤브레인 하이브리드화, 및 신호 감지를위한 표준 프로토콜의 수정된 버전을 사용하여 관심있는 유전자에 대한 검사를 할 수있는 방법을 보여주었다. 우리는 비디오와 함께 온라인으로 제공되는 모든하는 상세한 서면 관련된 단계에 대한 설명과 필요한 자료와 절차의 시각 데모를 보완.

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

Large-Scale Screens of Metagenomic Libraries

Metagenomic 도서관 대규모 화면

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

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Biology

8.5K 조회수.  MIT - Massachusetts Institute of Technology. 

비디오: 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.

전체 몽키 브레인에서 대규모 단백질 탐지에 대한 배치 Immunostaining

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

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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.

분리 및 성인 인간 내피 콜로니 형성 전구 세포의 대규모 확장

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.

대규모 Zebrafish 기반 생체내에서 작은 분자 화면

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.

Metagenomic 도서관에서 Cellulase 활동 Biomining에 대해 높은 처리량 화면

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.

컴퓨터를 이용한 대규모 시각화 및 췌장 아일릿 질량, 크기 유통과 건축의 부량

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.

Barcoded 효모 게놈 라이브러리의 경쟁력 화면

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.

대규모 SNP 유전자형 응용 프로그램에 대한 Infinium 분석

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.

대규모 유전자 넉다운에<em&gt; C. 엘레</em&gt; 강력한 기능 상실 표현형을 생성하는 dsRNA를 먹이는 라이브러리 사용

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.

민감한, 대규모 양적 대사 체학을위한 전략

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 : 효모 성장의 다 변수 정량화를위한 ​​대규모 방법

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