1) Zebrafish 에그 컬렉션 <ol><li> 이전에 화학 화면의 하루 오후, 10-20 zebrafish 사육 탱크를 설정합니다. 양식업 시스템에서 물을 각각의 탱크를 채우십시오. 물고기 그물을 사용하여 각각의 사육 수조 내부 컨테이너 한 성인 남성과 1-2 성인 여성을 전송할 수 있습니다. 구분선 서로의 남성과 여성의 생선을 분리합니다. 새장을 분류하고 그들을 통해 뚜껑을 넣어. </li><li> 화면의 아침, 사육 탱크에서 디바이더를 제거하고 짝짓기 zebrafish하실 수 있습니다. 다음 1 시간 과정 동안 수정된 계란 각 내부 컨테이너의 하단에 그리드를 통해 가을 수 있습니다. </li><li> 일시간 후, 영구 저장 탱크로 성인 zebrafish를 반환하는 내부 용기를 제거하고 플라스틱 차 스트레이너를 통해 각 사육 탱크에 물을 긴장하여 계란을 수집합니다. </li><li> 페트리 접시 위에 스트레이너를 반전하고 E3 매체를 포함하는 세척 병을 사용하여 페트리 접시에 계란을 내리려고 부드럽게 스트레이너를 씻어. </li><li> 불투명 나타나는 모든 unfertilized 계란은, 일회용 플라스틱 피펫을 사용하여 삭제해야합니다. 각 짝짓기 크로스는 약 200 배아를 얻을 것입니다. </li></ol> 2) 96 - 웰 플레이트에 배아를 Arraying <ol><li> 유리 파스퇴르 피펫을 사용하여 96 - 웰 플레이트의 각 우물에 E3 중간에 5 배아에 대한 전송합니다. </li><li> 소중히 배아를 치료하지 복용, 피펫 - 일단 태아가 96 - 웰 플레이트에 못하였 느니라 있으며, 12 채널 (300 μL 30)를 사용하여 우물 밖으로 가능한 한 많은 E3​​ 매체를 제거합니다. 12 채널 피펫을 사용하므로 같은 배아가 건조하지 못하게하는 것이 아니라 가능한 한 빨리 각각 0.5μg/ml kanamycin을 포함 E3 매체를 250 μL를 제공합니다. </li><li> 화합물을 추가할 때 그들이 원하는 무대를 도달할 때까지 28.5 ° C 배양기로 96 - 웰 플레이트 놔. </li></ol> 작은 분자 도서관 3) 전송 복합 전송이 로봇 전송 방법으로 자동화할 수 있지만, 우리는 수동 전송 방법을 설명합니다. <ol><li> 작은 분자 라이브러리는 일반적으로 10 MM 재고로 DMSO에 저장된 각각의 화합물로, 96 - 웰 형식으로 제공됩니다. 화합물이 추가 될 때 태아의 단계에 도달 약 60 분 전에, 작은 분자의 aliquots (소​​스 판)이있는 96 - 웰 플레이트에 원하는 숫자를 녹여. 소스 접시의 시리얼 또는 기타 식별 번호를 기록해 둡니다. 접시에 응축을 최소화하기 위해 해동은 Drierite을 (WA 하몬드 DRIERITE CO, 제니아, OH) 포함하는 건조 챔버에서 발생할 수 있습니다. </li><li> 간단히 멀티 웰 플레이트 어댑터가 장착된 탁상 원심 분리기에서 접시를 스핀 다운. </li><li> 소스 판에서 알루미늄 실링 테이프를 제거합니다. 12 채널 피펫을 사용하여 0.5 mm의 농도 (예를 들어, 10mM 재고 250 NL의 aliquots 시작하는 경우, 각 잘하는 DMSO의 4.75 μL 추가)하는 소스 접시에있는 성분을 희석. </li><li> 96 - 웰 플레이트에 배아 (받는 접시) 원하는 단계에 도달하면,를 포함하는받는 사람 접시에 소스 접시에서 화합물의 2.5μL (0.5mM)을 전송하는 12 채널 (2-20 μL) 피펫을 사용하여 배아. </li><li> 받는 사람 배아 접시에있는 소스 접시의 식별 번호를 기록합니다. 지금 뚜껑과 함께 배아와 화합물을 포함하는받는 사람 번호판 커버, 부드럽게 부드럽게 소용돌이 치는하여 번호판을 혼합하고, 28.5 ° C 배양기에서 그들을 놓으십시오. </li><li> 장기 저장을위한 -80 ° C 냉동고에 알루미늄 씰링 테이프 및 장소 사용되지 않은 작은 분자 (0.5 MM)를 포함하는 각 소스 판 표지. </li></ol> Phenotypes의 육안 검사에 의해 작은 분자의 효과에 대한 4) 심사 <ol><li> 이전 화면을 수행하는 &quot;히트&quot;를 구성하는 것이 무엇에 대한 구체적인 기준을 수립. </li><li> 개발에 원하는 시간에, 인큐베이터에서 화합물 - 처리 배아를 포함하는 96 - 웰 플레이트를 제거하고 stereomicroscope 아래의 각 우물을 확인합니다. 이러한 순환 패턴의 변화 등 미묘한 변화를보다 효율적으로 시각화 들어, 단계 - 대비 바뀌 현미경을 사용할 수 있습니다. 형광 현미경은 조직 특정 발기인에 따라 GFP 또는 DsRed 단백질의 표현의 섭동을 검사하는 데 사용할 수 있습니다. </li><li> 신속하게 5 배아 밖으로 적어도 3 소정의 &quot;히트&quot;표현형을 전시 어느 잘 대해 96 - 웰 플레이트를 검사합니다. 판 각 잠재적인 충돌의 잘 위치의 신원을 기록합니다. </li><li> 여러 복용 (1 음, 5 음, 10 μm의 및 50 μm의)의 화합물의 효과를 다시 테스트하여 잠재적인 충돌을 재확인. 각 복용량을 위해, 10 배아는 48 웰 플레이트 형식 E3 미디어 0.5 ML에서 테스트합니다. 다시 테스트를위한 화합물 이외의시기는 원래 심사의 동일해야합니다. 조회 elicited 표현형이 compoun의 다시 테스트를 재현 때 확인D </li><li> 화학 라이브러리에 작은 분자의 데이터베이스에서 히트 화합물을 식별합니다. </li></ol>

<ol>
	<li>Bayliss, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modulation+of+receptor+signaling+inhibits+regenerative+angiogenesis+in+adult+zebrafish.">Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish.</a> Nature chemical biology. 2, 265-273 (2006).</li><li>Burns, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+assay+for+small+molecules+that+modulate+zebrafish+embryonic+heart+rate.">High-throughput assay for small molecules that modulate zebrafish embryonic heart rate.</a> Nature chemical biology. 1, 263-264 (2005).</li><li>Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+structure-activity+relationship+study+of+dorsomorphin+analogues+identifies+selective+VEGF+and+BMP+inhibitors.">In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors.</a> ACS chemical biology. 5, 245-253 (2010).</li><li>Hong, C. C., Peterson, Q. P., Hong, J. Y., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artery/vein+specification+is+governed+by+opposing+phosphatidylinositol-3+kinase+and+MAP+kinase/ERK+signaling.">Artery/vein specification is governed by opposing phosphatidylinositol-3 kinase and MAP kinase/ERK signaling.</a> Curr Biol. 16, 1366-1372 (2006).</li><li>Milan, D. J., Peterson, T. A., Ruskin, J. N., Peterson, R. T., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+that+induce+repolarization+abnormalities+cause+bradycardia+in+zebrafish.">Drugs that induce repolarization abnormalities cause bradycardia in zebrafish.</a> Circulation. 107, 1355-1358 (2003).</li><li>Peterson, R. T., Link, B. A., Dowling, J. E., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+developmental+screens+reveal+the+logic+and+timing+of+vertebrate+development.">Small molecule developmental screens reveal the logic and timing of vertebrate development.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 12965-12969 (2000).</li><li>Yu, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsomorphin+inhibits+BMP+signals+required+for+embryogenesis+and+iron+metabolism.">Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.</a> Nature chemical biology. 4, 33-41 (2008).</li><li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nature reviews. 4, 35-44 (2005).</li><li>Stern, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecules+that+delay+S+phase+suppress+a+zebrafish+bmyb+mutant.">Small molecules that delay S phase suppress a zebrafish bmyb mutant.</a> Nature chemical biology. 1, 366-370 (2005).</li><li>Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+suppression+of+a+genetic+mutation+in+a+zebrafish+model+of+aortic+coarctation.">Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation.</a> Nature biotechnology. 22, 595-599 (2004).</li></ol>

관심 없음 충돌 선언하지 않습니다.

zebrafish 기반 화학 화면을 계획하는 경우 특히주의가 고려에서 표현형 및 표현형의 배경 율의 견고에 지불되어야합니다. 이것은 유도 표현형 화학 진압을 위해 스크린에 특히 중요합니다. 예를 들어, transgene의 열 - 충격 유도에 의한 표현형 들어, 표현형을 유도 조건 reproducibly 정확하게 unacceptably 높은 거짓 긍정적인 요금을 피하기 위해 화면을 시작하기 전에 밖으로 매핑해야합니다. 주의 계획과, ...

<ol><li> 원하는 유전자형의 성인 zebrafish의 20쌍 최소. </li><li> 이동식 내부 컨테이너와 디바이더 (수생 서식지)와 물고기 그물, 사육 탱크. </li><li> 배양 접시 (10cm). </li><li> 플라스틱 티 스트레이너. </li><li> 병 containg 배아 물을 씻으십시오. </li><li> 일회용 폴리에틸렌 전송 pipettes. </li><li> 폴리스티렌은 96 - 잘 왕복 하단의 분석 플레이트 (코닝 코스타, 로웰, MA). </li><li> 유리 파스퇴르 피펫 (피셔 과학). </li><li> 수동 피펫 펌프, 10 ML (벨 - 예술 제품, 페콰녹, NJ). </li><li> E3 배아 매체 : 0.003 % PTU을 (phenylthio - carbamide, 시그마, 세인트 루이스, MO)가 포함된 5 MM NaCl, 0.17 MM KCl, 0.33 MM CaCl 2, 0.33 MM MgSO 4. PTU는 E3 배아 미디어 1 L에서 0.3 - G PTU를 용해하여 10X 솔루션으로 준비하실 수 있습니다. PTU를 포함하는 솔루션은 알루미늄 호일로 덮고의 빛으로부터 보호되어야합니다. </li><li> 12 채널 pipettes, 2-20 μL (Eppendorf). </li><li> 12 채널 pipettes, 3에서 300 사이 μL (Eppendorf). </li><li> 일회용 피펫 폴리스티렌 분지, 50 ML (피셔 과학). </li><li> DMSO 10 MM 주식에서 96 못할거다 형식 못하였 느니라 구조적으로 다양한 화합물의 작은 분자 라이브러리입니다. 각 마스터 판은 96 잘 폴리 프로필렌 저장 플레이트 (코닝)로 aliquoted하고, -80 ° C에서 사용할 때까지 저장됩니다. </li><li> 알루미늄은 (Nunc, 로체스터, NY) 96 - 웰 플레이트에 대한 테이프를 씰링. </li><li> DMSO (시그마, 세인트 루이스, MO). </li><li> 기본 인큐베이터, 28.5 ° C (피셔 과학). </li><li> 전송 라이트베이스 (Leica 이크로 시스템즈, 배녹번, IL)로 Stereomicroscope. </li></ol>

large scale zebrafish-based in vivo small molecule screen

그들의 작은 배아 크기, 신속한 개발, 투명성, 생산력, 그리고 포유류에 수많은, 분자 및 생리 형태학의 유사성을 감안할 때, zebrafish는 강력한으로 등장했습니다<em&gt; 생체내에</em표현형 기반 약물 스크린 및 화학 유전 분석&gt; 플랫폼입니다. 여기, 우리는 zebrafish 배아를 사용하는 작은 분자의 대규모 심사 간단한 실용적인 방법을 보여줍니다.

Watch this Scientific Journal Video about Large Scale Zebrafish-Based In vivo Small Molecule Screen at JoVE.com

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish는 강력한으로 등장했습니다<em&gt; 생체내에</em표현형 기반 약물 스크린 및 화학 유전 분석&gt; 플랫폼입니다. 여기, 우리는 zebrafish 배아를 사용하는 작은 분자의 대규모 심사 간단한 실용적인 방법을 보여줍니다.

대규모 Zebrafish 기반 생체내에서 작은 분자 화면

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

large-scale-zebrafish-based-in-vivo-small-molecule-screen

Research

JoVE Journal

Biology

Large Scale Zebrafish-Based In vivo Small Molecule Screen

13.4K 조회수.  Vanderbilt University School of Medicine. Zebrafish는 강력한으로 등장했습니다 생체내에 플랫폼입니다. 여기, 우리는 zebrafish 배아를 사용하는 작은 분자의 대규모 심사 간단한 실용적인 방법을 보여줍니다.

비디오: Large Scale Zebrafish-Based In vivo Small Molecule Screen

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic 도서관 대규모 화면

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

연구

생물학

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 ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

유전자 표현 기계의 기능 Assays을위한 소규모 핵 추출물

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

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 ...

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 ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish의 배아를 사용하여 높은 처리량 접근 수동 작은 분자 화면

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 &#176;C for 30 min. Thereafter, the milk is cooled down to 45 &#176;C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 &#176;C and 45 &#176;C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 &#176;C for 30 min, and subsequently cooled to 45 &#176;C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 &#176;C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture.

생균제 발효 식품의 소규모 제조를위한 신규 생산 프로토콜

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter ...

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, ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

헬리안투스 아누스의 대규모 열화상 스크리닝에 의한 새로운 식물 증발 규제 기관 식별

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

대규모 후생유전학 연구를 위한 반자동 ChIP-Seq 절차

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...