游泳池的设计和寡复苏<ol><li>第一步是要研究设计的前体mRNA池。这是可以做到使用UCSC基因组浏览器和下载的特定基因，拼接路口，或其他感兴趣的领域。 </li><li>一旦利益的窗户已被选中，在它们之间的瓷砖计算使用下列条件：读取的长度应为30与10核苷酸重叠，因此​​每个寡核苷酸转移从之前的20个核苷酸核苷酸。 *寡重叠，应增加在接近的剪接位点，以确保足够的覆盖范围;增加10至20个核苷酸的重叠，可以做到这一点。 </li><li>侧翼每个与通用引物序列，这将是用来放大池下游30个核苷酸的寡核苷酸。应提交安捷伦，将一个自定义的寡核苷酸芯片印刷序列平铺寡订单。 </li><li>要恢复从芯片表面的DNA寡核苷酸开始放置在阵列杂交室阵列封面幻灯片和幻灯片上轻轻移液500μL的dh 2 </li><li>夹心数组封面幻灯片上，照顾，以避免气泡，可以破坏的过程。可以肯定的数组正面朝下放置，以便与寡核苷酸的一边是接触水面。一个好的经验法则是“安捷伦触及安捷伦”，意思是阵列上的安捷伦标签面临着封面上的幻灯片安捷伦标签</li><li>关闭杂交室和旋转在99℃过夜，在杂交炉</li><li>翌日，小心地取出数组，并从会议厅制定的水，它现在包含寡核苷酸阵列的表面解放。这是寡核苷酸池</li><li>在50％幅度将在1.5 mL Eppendorf管的游泳池和超声池3-5个脉冲</li><li>接下来，由低循环PCR扩增池，使用的末尾加上一个T7号标记的通用引物。对于第一轮，变性94 ° C 1分钟，55℃退火20秒，1分钟拉长为72 ° C。随后的几轮，做10秒，20秒，10秒，在每个相应的温度，5分钟，最后延伸72 ° C。由于寡恢复的效率取决于放大池所需的周期数，可能需要尝试多个周期的数字。要小心不要过度放大的游泳池，这是由丙烯酰胺凝胶涂乐队标志着。 PCR扩增产物可存放在4℃直至需要。 </li><li>在寡准备的最后一步是利用Ambion公司MEGAshortscript™套件转录RNA。 *以下协议改编自Ambion公司<ol class="lalpha"><li>解冻的T7 10X反应缓冲液，在室温下4个核苷酸的解决方案和水，同时保持冰的T7酶组合</li><li>组装反应混合物在室温下的无RNase的离心管。反应缓冲液的组成部分可以使模板DNA沉淀，如果反应是在冰上混合。 </li><li>按照以下顺序，吸管3μL无核酸水，10X反应缓冲液，每75毫米NTP解决方案2μL，5μL模板DNA（PCR扩增寡核苷酸池）和2μL的T7酶，一个终体积20μL </li><li>管轻轻一抖混合反应，然后简要离心收集管底部的混合物。 </li><li>在热循环反应在37℃2小时。孵化时间将依赖模板，因此它可能是必要的使用时间课程的实验，以确定获得最大的收益率孵育时间</li><li> 2小时后，取出一个额外的15分钟的模板DNA加入1μL涡轮DNA酶反应和孵化，在37 ° C </li><li>终止反应和恢复酚/氯仿抽提和乙醇沉淀的RNA。 <ol class="lalpha"><li>为20μL的反应体积，加115μL的dh 2 O和15μL3 M醋酸钠的反应。 </li><li>调匀再加入75μL酸性苯酚和75μL氯仿。由涡旋混合溶液，然后在室温下13000转一分钟的离心。水层转移到一个新的管和重复提取</li><li>转移到一个新的管水层，并添加150μL氯仿。涡旋和以前一样的混合物离心机。水层转移到一个新的管。 </li></ol></li><li>两卷的100％的冷乙醇添加水层和混合以及收回的RNA。冰寒的混合物，最少为15分钟，在-20 ° C，然后离心机在4 ° C时最高速度在15分钟沉淀的RNA。小心取出上清液，和105的0.1倍的Tris - EDTA（TE）缓冲液重悬沉淀.. </li></ol></li><li>这是很难删除所有从T7套件反应的游离核苷酸，最准确的结果，量化的RNA使用RiboGreen的。 <ol class="lalpha"><li>要开始定量，先稀足够的1X TE，2300μL+50μL/sample。 </li><li>接下来，RiboGreen。您将需要1000μL，50μL/样本。对于大范围的样品（1微克/毫升至20毫微克/毫升）混合在1x TE RiboGreen 1:200;低范围样本（50毫微克/毫升为1毫微克/毫升）混合在1x TE RiboGreen 1:2000 ;移液器到每一个不透明板以及50μLRiboGreen解决方案，开始与A1和向下移动，而不是跨</li><li>稀释到2微克/毫升高范围的样品或为100 ng /μL低范围的样品RNA原液</li><li>利用股票的解决方案，创建标准曲线由六个随后1:2稀释原来的2微克/毫升或100毫微克/毫升股票;移液器的股票240到μL的PCR条管。在余下的7管，吸液管120μL的1X TE。取120μLRNA的股票，混合管2。从管2 120μL和混合管3。重复，直到管7。管8只会有TE和将您的空白。吸取50μL的标准曲线的解决方案不透明板，A1：H2（两次运行曲线） </li><li>在一个新的管中，45μL的TE 5μL的寡核苷酸池混合，并添加所有50μL到包含50μLRiboGreen良好。 </li><li>使用酶标仪设置本机读取从顶部的荧光，后一分钟的组合激发波长485 nm，发射波长为530 nm读板</li><li>使用标准曲线生成量化回收的RNA，应储存在-20 ° C </li></ol></li></ol>免疫共沉淀的寡核苷酸池与利益的RNP <ol><li>免疫共沉淀开始之前，准备A和蛋白G磁珠从Invitrogen公司以1:1的比例混合，最终成交量的50％μL反应的蛋白质。例如，对于两种反应中，您将添加50μL的每个珠子。 </li><li>使用一个像这样一个自Novagen磁分离架举行到位的珠子，而你去除上清，洗珠与冷的1X PBS等体积的两倍</li><li>第二次洗涤后，在同等体积的冷1X PBS重悬珠，并添加2％反应的抗体吴。这一数额可能会有所不同，根据抗体，因此试验是必要的，以找到最佳的条件。 </li><li>抗体/珠的混合物过夜孵育4 ° C的一个旋转平台</li><li>上午，添加2微克每超声酵母总RNA的反应，以阻止非特异性结合，和一个额外的30分钟旋转4 ° C。 </li><li>使用磁机架举行的珠子，去除上清，洗珠两次用冷的1X PBS，留在管内的第二次洗的PBS。 </li><li>取出新的管子，每个反应和每管分装50μL珠混合。让珠坐，同时准备一个主结构</li><li>每个反应中添加200毫微克的寡核苷酸池，保护引物摩尔比，超声酵母总RNA 2微克，20μLHeLa细胞的核提取物（这是RNP源），和缓冲区发送带来的音量高达120微升。保护引物的通用引物RNA的版本。订购转录模板在末尾加上一个T7号标记的通用引物，然后抄写使用MEGAshortscript™套件模板的RNA。 </li><li>从珠分装和主结构在120μL重悬珠取出上清液。在4 ° C为1-2小时，岩石的反应。 </li><li>孵化后，每个反应，一个“总”的RNA样品中删除一个小等份（包括珠） </li><li>将其余磁选立场的反应，去除上清。虽然是没有必要的分析样品下游流的，它可能是有用的保存的。 </li><li>洗磁珠冷1X PBS 50μL的1-2倍。绑定珠抗体的RNA是“知识产权”的样本。 </li><li>在1％的0.1倍TE缓冲液中的SDS 50μL重悬的“总”和“IP”珠和样品的温度在65℃，15分钟洗脱RNA。 </li><li>去除上清，它现在包含以前绑定的珠子和干净的酚/氯仿抽提乙醇沉淀的RNA。干颗粒，并在50μL的0.1倍TE缓冲液重悬。 </li></ol>合作IP样本的逆转录和扩增准备aminoallyl标签<ol><li>接着，RNA样品，必须反转录和PCR扩增。逆转录反应，使用反向通用引物。 </li><li>对于每个样本，混合1μL开始反向通用引物，1μL模板的RNA（免疫反应）和10μLDH 2 O。热的反应，在70 ° C 5分钟，然后寒意在冰上或保持在4 ° C。 </li><li>当样品冷却，使以下主结构。对于每一个反应，结合10毫米dNTPs 4μL，2μL10X Arrayscript缓冲区，1μLRNase抑制剂，和1μL反转录酶。调匀，加入8μL主结构每个逆转录反应。 </li><li>使用PCR仪，样品在42 ° C孵育2小时，95 ° C 5分钟，然后4 ° C，直到样品需要放大。 </li><li>反向转录后的RNA样品，PCR扩增使用的通用引物，含有T7启动标记的引物之一。放大池所需的周期数将取决于合作IP的效率，因此它可能是需要尝试多个周期。每个反应需要5μL10倍的Taq反应缓冲液，0.2μL每个引物（100毫米），cDNA模板1μL（逆转录反应），10毫米dNTPs 1μL，42.2μLDH 2 O， 0.4μLTaq酶。 开始3分钟，94℃孵育样品的PCR。接下来，每个周期内，变性94 ° C 45秒，退火55 ° 30秒，在72 ° C和拉长最终伸长率在72℃10分钟，一分钟。 </li><li>下一步，抄写样品​​再次使用MEGAshortscript™套件，然而，这一次加2μLaminoallyl双绞线，而不是T7双绞线。 aminoallyl双绞线将用于标签的Cy染料的RNA。 </li><li>酚/氯仿和乙醇沉淀的样品。不要使用Glycoblue或醋酸铵，这两者都可能干扰样品的杂交到阵列。干颗粒和重悬在4.5μL0.1 M的碳酸钠。 </li></ol>氨基烯丙基标记的RNA用Cy染料<ol><li>要开始的标签协议的荧光染料，Cy3和Cy5溶解于45μL二甲基亚砜，。为了避免光线，在黑暗中管包裹在铝箔和储存在-20 ° C </li><li>添加2.5μL无核酸酶水RNA样品调匀</li><li>接下来，添加3μL的“总”的样本，Cy3标记，与3μL的Cy5的“知识产权”样本。在黑暗中孵育样品在室温下1小时，CY - 3样品会变成红色或粉红色，而CY - 5会变成蓝色。 </li><li>终止反应，加入6 4米羟胺盐酸UL，每个样品，调匀，自旋简要。 15分钟在室温下孵育。 </li><li>净化酚/氯仿抽提，乙醇沉淀和重新悬浮颗粒50μL的0.1倍TE标记的样品</li></ol>杂交样品芯片使用安捷伦阵列杂交试剂盒<ol><li>首先，轻轻混匀50μL封闭液的10倍，140核酸无H2 O，Cy3标记的“总”池，25μL的Cy5的标记为“知识产权”池25μL，10μL的微升杂交25X碎片缓冲区和250μL的2倍的杂交缓冲液，同时注意避免引入气泡。离心样品简单地收集在试管底部的解决方案，同时也减少气泡。 </li><li>将O形圈在杂交室的封面幻灯片，并仔细移液器到幻灯片的解决方案。 </li><li>将“三明治”的解决方案的封面幻灯片上的探测阵列。请记住，东方的阵列幻灯片，使阵列上的安捷伦标签面临着封面上的幻灯片安捷伦标签。 </li><li>夹在杂交室幻灯片/阵列三明治和旋转50 ° C时为3小时。 </li><li>而阵列旋转，请在50 mL锥形管50毫升的解决方案：一个圆锥形充满50毫升生署2 O两个1X SCC的缓冲溶液中，每一个单独的管，和一个2倍SCC的缓冲区。 </li><li>三个小时的潜伏期后，小心地松开阵列/幻灯片夹层，放置于50 ml含有2倍的SCC的缓冲区锥形。使用镊子，轻轻分开的封面幻灯片阵列拉出封面幻灯片。要小心，不要触摸幻灯片拉出阵列。关闭锥形，轻轻地反转60秒。 </li><li>使用镊子，转移到1倍SCC解决方案的阵列，帽筒，轻轻倒置1分钟，然后转移到阵列中的第二1X SCC解决方案，并再次轻轻地反转一分钟。 </li><li>最后，转移到阵列中的DH 2 O管，轻轻地反转30秒。 </li><li>干的幻灯片的边缘上放置一个Kimwipe数组。继续旋转的阵列擦拭确保不会破坏含有寡核苷酸的一面。 </li><li>将干阵列一个空的50毫升锥形管和微阵列扫描仪扫描</li></ol>解读基因芯片<ol><li>芯片扫描仪将创建一个数组的TIF图像。使用网格文件（由安捷伦提供的），这是您的特定池独特的形象，通过安捷伦的特征提取软件运行。 </li><li>安捷伦软件将解释每个功能的信号，创建一个文件，这是一个Perl脚本解析。在每个功能的信号是归在该通道的总强度。例如在一个特定功能的绿色通道，个别功能的绿色通道的强度除以总强度以上所有功能的绿色通道。最后的输出是在阵列上的每个功能的绿色通道，红色通道的日志比例。 </li><li>从芯片的寡核苷酸池中的数据可以被重新映射到基因组得到一个图片在哪里，以及如何强烈，利益的RNP约束。 </li></ol> 图1。实验原理图 <img src="/files/ftp_upload/1622/1622fig1a.jpg" alt="图1a" /> A.切片方案。选择和下载利益的前体mRNA序列后，通过选择面积平铺在30 10个核苷酸的增量转向核苷酸窗口。通用引物结合位点侧翼每边的窗口。 <img src="/files/ftp_upload/1622/1622fig1b.jpg" alt="图1b" /> B.合成阵列。应提交安捷伦，将一个自定义的寡核苷酸芯片印刷序列平铺寡订单。 <img src="/files/ftp_upload/1622/1622fig1c.jpg" alt="图1c" /> C.免疫共沉淀。煮沸后关闭阵列的寡核苷酸，在HeLa细胞的核提取物孵育，然后加入准备对利益的RNP抗体的磁珠提取。标签用Cy3寡核苷酸池，并用Cy5绑定的IP地址池，适用于探测阵列和扫描。 图2。注释基因组和浓缩数据分析。 <img src="/files/ftp_upload/1622/1622fig2.jpg" alt="图2" /> A.平均得分从数组中的数据，在每个基因组的协调基地，十个日志和地图转换得分给定的坐标。这个平均步的一个例证是3个重叠的30 nt的寡核苷酸2分，4，0.5，平均每10 NT窗口浓缩得分作图以上。可以可视化USCS基因组浏览器中使用自定义跟踪选定的基因组区域。例如浏览器窗口是在CLTA基因，基因的功能（外显子/内含子/选择性剪接等），沿底部，并从客运大楼的约束寡核苷酸日志平均富集分数由暗红色的竖线代表。客运大楼的强烈结合在polypyrimidine道外显子2上游。

执行此过程时，重要的是记住，池创建灵活和开放的修改，在RNA或蛋白质水平。在RNA水平同源地区，疾病基因突变，多态性，或随机突变可以被引入到寡核苷酸序列。在蛋白质水平的RNA结合因子可以修改的磷酸化或其他翻译后修饰。此外，具有约束力的环境可以被操纵，以增加或减少交互或与RNA结合蛋白的利益竞争的其他因素的水平。

a rapid high-throughput method for mapping ribonucleoproteins (rnps) on human pre-mrna

测序合作与RNA结合蛋白免疫沉淀（CO - IP）增加表明装订位置的RNA拼接的认识，往往影响一个剪接因子的功能。然而，与任何抽样策略确定绑定到剪接因子一种RNA的机会是其细胞丰度成正比。我们已经开发出一种在体外培养的方法的前体mRNA上，否则瞬态测量结合的特异性的小说。这种方法采用了专门设计的寡核苷酸池，跨内含子，外显子剪接路口，或其他前体mRNA的瓷砖。池是受到某种分子选择。在这里，我们展示了通过分离成一个绑定和非绑定小数寡核苷酸的方法，利用两种颜色的数组战略，在绑定的分数记录每一个寡核苷酸的富集。阵列中的数据能够识别特定序列和结构determinates核蛋白（RNP）的前体mRNA结合生成高分辨率地图。这种方法具有独特的优势是它的能力，以避免对当前的IP和SELEX技术与相关的基因的抽样偏差，池是专门设计和合成的前体mRNA序列。寡核苷酸池的灵活性是另外一大优势，因为实验者选择哪个地区的研究和瓷砖跨他们的个性化需求，剪裁池。使用这种技术，可以检测约束力的大规模成一个功能的剪接记者的克隆库，并确定是在所包含的部分丰富的寡核苷酸多态性或突变的影响。这小说在体外高分辨率测绘计划提供了一个独特的方式来研究瞬态mRNA前体的物种，其丰度低，使得他们难以电流在体内的技术研究与RNP的相互作用。

Watch this Scientific Journal Video about A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA at JoVE.com

A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

由于mRNA前体的瞬态的性质，它可以很难分离和研究<em&gt;在体内</em&gt;。在这里，我们提出了一种新<em&gt;在体外</em&gt;调查使用一种人工合成的寡核苷酸池的RNA -蛋白质相互作用的方法，整个选定区域的前体mRNA的瓷砖。

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

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A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

11.7K 次观看.  Brown University. 由于mRNA前体的瞬态的性质，它可以很难分离和研究在体内。在这里，我们提出了一种新在体外调查使用一种人工合成的寡核苷酸池的RNA -蛋白质相互作用的方法，整个选定区域的前体mRNA的瓷砖。

视频: A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

科研

生物学

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

冰帽：为发展方法拟南芥和番茄植株在96孔板高通量的基因分型

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...