这项工作是由来自国家科学基金会（批准号MCB - 0447750）的资助。

1。准备与琼脂生长介质的冰帽育苗板熔融生长介质（0.5X Murashige和Skoog（MS）的基础盐的混合物，2毫米Morpholinoethanesulfonic酸（MES），琼脂0.6％（W / V），pH值5.7）免除使用MicroFill微孔板饮水机进入蒸压幼苗板。一公升的媒体是需要每18个育苗板。 <ol><li>准备螺杆顶部的盖子和高压灭菌20分钟一公升玻璃瓶生长介质。 </li><li>高压灭菌器一公升的蒸馏水在一个玻璃瓶螺杆顶盖。同时也反应釜空育苗铝箔板。 </li><li>孵化器集传输蒸压媒体和水，在65 ° C。这个温度将保持在熔融状态下的生长介质。 </li><li>高压灭菌后，取出铝箔和层流罩干育苗板。板必须完全干透后再进行，使胶带包装胶带完全密封板井底部的孔。 </li><li>实验室工作台上用95％的乙醇消毒。 </li><li> 95％的乙醇浸泡在透明的塑料盖幼苗板来净化。从乙醇和干燥的空气层流罩盖。不要高压灭菌器的塑料盖，消毒，因为他们会融化在高压釜。 </li><li>封箱胶带每个育苗盘的底部，并牢牢密封使用辊工具。 </li><li>将定为65 ° C水浴中熔融生长介质和无菌水的瓶子</li><li>将一瓶含有70％的乙醇，在室温MicroFill机和运行两次消毒MicroFill机管道吹扫周期。 </li><li>附加MicroFill机用无菌水的瓶子。清洗循环运行六次热烈的管道，并明确了系统的乙醇。整个此过程中，一瓶水应该保持在65℃水浴。 </li><li>完成后，用无菌水清洗，立即将一瓶熔融生长介质的MicroFill机器。清除周期运行两次，清除管道蒸馏水。生长介质的瓶子应保持在65℃水浴的整个过程。 </li><li>免除到每个育苗盘井450微升的生长介质。如此迅速的生长介质，不巩固MicroFill机管道的。 </li><li>当所有的苗子板已经排满，立即附上一瓶65℃无菌水MicroFill机，清洗循环运行12次，清理管道。一瓶水应保持在65℃水浴的整个过程。 </li><li>如果MicroFill机无法使用，也可以准备育苗板手移液使用多通道移液器的密封板熔化琼脂。 </li><li>检查每个育苗盘吨的水井确保生长介质不泄漏任何水井。吸管手成单个井的熔融生长介质需要。 </li><li>每块板带有明显的塑料盖盖，以及允许增长的媒体，以巩固在室温下的幼苗板。栈板和包装铝箔用于存储在4 ° C商店板倒置，以防止在储存过程中的井积水。 </li></ol> 2。到冰帽育苗板和发​​芽幼苗播种我们曾描述为一个半自动化的方法，使用种子装载设备（2）成苗期板配药拟南芥种子。虽然这种设备可以有用的部分批次的种子，我们发现，在不同批次的拟南芥种子显示种子大小的变异限制该设备的一般工具。我们在发展替代种子分配的自动化方法的过程中，BU吨，我们目前已经发现，配药种子到幼苗板的最有效的策略是通过手工执行此步骤如下所述。 <ol><li>干种子撒到滤纸滤纸。 </li><li> 95％的乙醇，使所有的种子都盖用乙醇免除到种子。这种治疗方法将表面消毒的种子。 </li><li>允许种子，空气干燥。 </li><li>转移到个别种子育苗使用修改后的一次性玻璃巴斯德吸管板块的井。巴斯德吸管密封燃烧的提示。由此产生的玻璃尖蘸轻轻抚摸琼脂培养基，它允许一个单一的种子很容易拿起和转移到琼脂表面的育苗盘井。一个双头版本，这个种子的电镀工具也可以使用，以提高吞吐量。 </li><li>每个育苗板盖上一个透明的塑料盖和密封微孔手术磁带。包装叠板铝箔和拟南芥种子储存于4 ° C的4天，分层，种子和同步发芽。番茄种子应保存在12 ° C为4天，在黑暗中。 </li><li>在荧光灯下放置在18 °〜20 ° C至启动萌发的幼苗板。 </li></ol> 3。将育苗板冰帽喷泉四天后的幼苗在轻板，转让幼苗板冰帽的喷泉。 拟南芥幼苗通常在10至14天的冰盖喷泉左边。 <ol><li>自定义机架结构已内放置一个塑料储物盒上放置一个cookie表组装冰帽喷泉。使用水平螺母，调整Cookie表的水平。放置在储物盒的潜水泵，并使用一个弹簧加载CLA重视输出软管泵侧的Cookie表MP。在植物生长室或生长室的架子上组装冰帽的喷泉，使表面的冰帽喷泉收到直接的荧光灯照明。 </li><li> 3个部分1部分自来水蒸馏水的混合物填充冰帽喷泉。应加水喷泉，潜水泵，直到水位以下几厘米。标记的塑料储存，使用实验室磁带盒和标记笔的最终水位。 </li><li>准备加入3/32-inch直径不锈钢球以及每根板根板。 </li><li>不锈钢球可以被添加到根板，使用球配药设备 1自定义。此设备是由放置在96孔PCR板的表面的铝箔单张纸和使用标记笔标记每口井的中心位置上的铝箔表面。这种箔显着的表，然后放置在一个额外的6铝板顶部IL和缠盖子表面光滑，从使用的枪头中。如木叉叉的一个利器，然后用于创建一个大到足以容纳一个单一的金属球的96个职位，每年的草皮。为了填补这个球配药设备，金属球过剩是浇过的设备，并轻轻摇匀，去除多余的球。根板，然后放置在配药设备，这是翻转下降到每根板以及一球的顶部。 </li><li>新增340微升3个部分1部分自来水蒸馏水的混合物以及每根使用的MicroFill机板。确保配药水根板，已添加钢球。 </li><li>剥离胶膜关闭的育苗板的底部，从板块中删除透明的塑料盖。每个育苗板放置在根包含一个金属球和水板和安全两大板块一起使用两个弹性海ř乐队。 </li><li>在冰帽的喷泉广场的叠冰帽板。板块在冰帽喷泉，透明的塑料盖，不应使用。检查，以确保的育苗盘，以及A - 01是同根板A - 01对齐。 </li><li>堆叠的幼苗/根板应留在冰帽的喷泉，直到大多数的幼苗根组织已渗透到根板。 拟南芥幼苗在冰帽喷泉理想的生长温度是CA 。 18 ° C。 Cookie表的水位应这样的水可以流入根板井，但不能如此之深，在育苗盘秧苗被淹没。如果水位不正确，您将需要得到一个正确的深度不同的Cookie表。 </li><li>定期添加蒸馏水，冰帽的喷泉，保持原有的水位。加入纯蒸馏水的喷泉，你将取代液体因蒸发而损失的不会改变的喷泉水的矿物成分。水位应在冰帽的喷泉每天检查。 </li></ol> 4。嘉实根组织根组织是冻结在根盘的水，然后从根盘育苗板，脱皮收获。 <ol><li>收获根组织的前一天，取出从冰帽喷泉叠冰帽板。 </li><li>插入三个堆叠的幼苗和根板井之间的木串。肉串的目的是提高准备冻结过程中根板孔的育苗板的笔尖。 </li><li>允许插入一夜之间站在灯光下的木串叠冰帽板。此潜伏期可根板，这有利于随后冻结和板块分离过程中的液体轻微蒸发。 </li><li>在组织收获的一天，准备冻结浴。一个96孔的金属热块放置在干冰和95％乙醇的混合物的耐热盘。允许热块温度平衡为CA。 20分钟。放置在顶部的某些类型，如手套，所以，温度低，不损伤的实验台表面高压灭菌器的绝缘材料的耐热盘。 </li><li>将堆叠的冰帽板，木串到冷冻的热块，并培育5分钟。在此期间，在根盘的水会冻结。在育苗盘的幼苗不冻结在此期间的待遇，并保持完全可行的。 </li><li>删除堆叠的冰帽板和热块放置在室温下的实验室工作台上。删除的松紧带和叠板木串。坚决按顶部的育苗盘“破解”板块。然后小心剥离根盘和育苗板AP艺术。 </li><li>允许水根板在室温下解冻。解冻可以加快孵化根板热块设置在25 ° C。 </li></ol> 5。存放在低温，黑暗中的幼苗板组织收获，育苗板可以存储在黑暗中低温将保持幼苗的可行性，同时进行基因型分析。苗可存储在几个星期，这些温度而不影响可行性。 <ol><li>组织收获后，用胶带密封的育苗板下部。 </li><li>顶部的育苗盘放在一个透明的塑料盖子和安全与微孔磁带。 </li><li>堆栈几个育苗板，包装铝箔。 </li><li> 拟南芥幼苗，板放置在4 ° C 。对于番茄幼苗，放置在12 ° C的板一旦基因分型是完整的，SEedlings可以从这样低的温度孵化，并移植到土壤，如下所述。 </li></ol> 6。 DNA的提取可以很容易地从根组织样本中提取DNA适用于PCR反应。从拟南芥根的组织样本回收的总DNA的产量是CA。平均2 400纳克。 <ol><li>确保水根板已经完全解冻后再进行DNA提取过程。检查，以确定是否有任何水井水平均大大低于板块。手移液器蒸馏水水成井，需要更多的水。 </li><li>使用MicroFill机添加25微升至每根板以及一个的Tris - EDTA溶液（500毫米三，pH值为8; 50 mM的EDTA，PH8）。 </li><li>使用热封铝箔及热封机板根密封的水井。 </li><li> GenoGrinder机放在与密封根板密封端板朝下。这个板块的方向提供了最优珠的运动，并防止坚持在井珠。为3分半钟摇板在每分钟1350招在GenoGrinder。 </li><li>板块转移至离心微孔​​板运营商和10分钟旋转3500转板块在4 ° C至颗粒煤粉根组织。 </li><li>稀释后的提取物含有基因组DNA可直接用于PCR反应。通常情况下，将增加2微升的这种DNA提取，PCR反应。 </li><li>对于存储的DNA样本，密封微孔板使用粘合剂包装胶带和地点在零下20 ° C。 DNA样本可以保存几个月，在质量上没有明显减少。 </li></ol> 7。与期望基因型幼苗转移到土壤基因型分析完成后，苗所需的基因型可以从幼苗转移土板。 <ol><li>从冷库中取出的育苗板，并准备与水饱和土混合物盆。 </li><li>要删除个人从育苗盘育苗，使用镊子，允许一抢而破碎的苗子琼脂插头。抓住镊子琼脂插头，小心不要损坏苗木的育苗板轻轻滑出琼脂插头。或者，可以用加压空气，轻轻地拍琼脂插件和幼苗以及。在井底部的小孔施加压力，直到弹出插件到实验室台上。 </li><li>准备在土壤中的琼脂插头大小的小孔。在土壤中的琼脂插件和包琼脂插头周围潮湿的土壤，以确保在土壤中的幼苗。不要试图删除任何来自各地的苗的琼脂。 </li><li>放置在一个标准的增长条件下的增长空间，移植苗。盖上一个塑料盖锅移植后的头两天。随后取下盖子，并继续使用标准条件下生长的植物。 </li></ol> 8。代表性的成果： 使用冰帽系统种植拟南芥的一个例子是如图4A所示。两个星期，这些树苗已在冰帽的喷泉照片的拍摄时，准备组织收获。从这些相同的幼苗根组织如图4B所示。通常是从拟南芥根组织样本中提取DNA的总金额在100纳克范围为 2 700吴。这是基于只有一个部分粗根提取物的使用收益计算。因为只有CA。 10％的粗根提取物通常是使用DNA提取过程中，它有可能获得显着更多的基因组DNA，保留更多的粗提取物的下游分析。基因组DNA的总量得到使用冰第程序是足够的执行数百个PCR 反应 2 。 <img alt="图1" src="/files/ftp_upload/3280/3280fig1.jpg" /> 图1：冰帽过程流程图。一）育苗板含有琼脂生长介质上的幼苗发芽。根穿透琼脂和发展朝盘底。孔在板孔的底部密封胶膜。二）育苗板堆放在上面的根板含有水和一个金属球。根退出的育苗盘井底部的孔，并成长为根盘井。三）木制串之间插入的育苗盘和根板的前一天堆放的冰帽板放置在干冰/乙醇浴thermoblock。 D）一旦根盘的水已冻结了坚实的，育苗板剥离从根盘，产生96个组织样本根板和96个可行的苗育苗盘。木串促进两个板块的分离，在这一步。 <img alt="图2" src="/files/ftp_upload/3280/3280fig2.jpg" /> 图2：苗盘和根盘的照片。 A）育苗板根板分开看。 B）育苗板根盘上堆放。可以看出，在根盘井钢DNA提取使用的球。弹性带一起使用，以确保这两个板块。 <img alt="图3" src="/files/ftp_upload/3280/3280fig3.jpg" /> 图3：第冰喷泉。第冰喷泉是用来维持一个恒定水位，在井根板顶部的精确高度。这种连续的洒水系统，确保根板井的水不会成为耗尽，由于蒸发或蒸腾。一）一个家庭作出的机架支持堆叠的冰帽板坐的Cookie表。 B）1“坚果，提供了精确调整Cookie表的水平，使一个统一的水深是实现整个表面喷泉的的手段之一的特写视图C）此图片显示的所有部分需要构建自制的机架冰帽喷泉D）组装冰帽的喷泉。潜水喷泉泵不断将水从下水库，在于自制上的Cookie表机架一个弹簧钳是用来连接软管Cookie表的边缘。 <img alt="图4" src="/files/ftp_upload/3280/3280fig4.jpg" /> 图4： 拟南芥幼苗生长过程中使用的冰帽。一）顶视图向下看苗盘成两个井。每口井包含拟南芥幼苗。二）两个井根盘的侧视图。根可看到扭曲周围的水井内表面。在这幅画中的幼苗已在冰帽喷泉CA。两个星期，处于成长阶段，组织收获通常会。

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	<li>Clark, K. A., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+An+improved+high-throughput+method+for+generating+tissue+samples+in+96-well+format+for+plant+genotyping+(Ice-Cap+2.0).">Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0).</a> Plant Methods. 3, 8-8 (2007).</li><li>Krysan, P. I. c. e. -. c. a. p. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+capturing+plant+tissue+samples+for+genotype+analysis.">A high-throughput method for capturing plant tissue samples for genotype analysis.</a> Plant Physiol. 135, 1162-1169 (2004).</li><li>Jander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+map-based+cloning+in+the+post-genome+era.">Arabidopsis map-based cloning in the post-genome era.</a> Plant Physiol. 129, 440-450 (2002).</li><li>Liljegren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHATTERPROOF+MADS-box+genes+control+seed+dispersal+in+Arabidopsis.">SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis.</a> Nature. 404, 766-770 (2000).</li><li>Buechel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+A-type+ARABIDOPSIS+RESPONSE+REGULATORS+in+meristem+maintenance+and+regeneration.">Role of A-type ARABIDOPSIS RESPONSE REGULATORS in meristem maintenance and regeneration.</a> Eur. J. Cell. Biol. 89, 279-284 (2010).</li><li>Krysan, P. J., Jester, P. J., Gottwald, J. R., Sussman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mitogen-activated+protein+kinase+kinase+kinase+gene+family+encodes+essential+positive+regulators+of+cytokinesis.">An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis.</a> Plant Cell. 14, 1109-1120 (2002).</li><li>Bush, S. M., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iTILLING:+a+personalized+approach+to+the+identification+of+induced+mutations+in+Arabidopsis.">iTILLING: a personalized approach to the identification of induced mutations in Arabidopsis.</a> Plant Physiol. 154, 25-35 (2010).</li><li>Comai, L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING:+practical+single-nucleotide+mutation+discovery.">TILLING: practical single-nucleotide mutation discovery.</a> Plant J. 45, 684-694 (2006).</li><li>Henikoff, S., Till, B. J., Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING.+Traditional+mutagenesis+meets+functional+genomics.">TILLING. Traditional mutagenesis meets functional genomics.</a> Plant Physiol. 135, 630-636 (2004).</li><li>McCallum, C. M., Comai, L., Greene, E. A., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+induced+local+lesions+IN+genomes+(TILLING)+for+plant+functional+genomics.">Targeting induced local lesions IN genomes (TILLING) for plant functional genomics.</a> Plant Physiol. 123, 439-442 (2000).</li></ol>

没有利益冲突的声明。

这里介绍的方法，使冰帽一位科学家从数以百计，甚至数以千计的个别工厂，在某一天收集组织样本，并有效地从这些样本中提取基因组DNA用于基因分型反应。冰帽方法给予科学家个人的能力基因型数以千计的幼苗，在很短的时间内，有潜力，以促进遗传实验，否则不会实际执行数。下面提供了几个例子。 <ol><li> 遗传图谱 。如果研究人员能够确定沿3号染色体的一个特定的间隔内的重组事件，可以加快规模精细的遗传图谱。如果这个时间间隔涉及到一个很短的遗传距离，然后将重组事件比较少见的分离映射的人口。出于这个原因，它可能是必要的屏幕数以千计的个别工厂找到所需的重新的combinant个人。这个过程可以通过使用冰帽的步伐大大加快。即使在全基因组重测序的时代，它仍然是必要的直接体现，一个给定的突变是直接负责为有兴趣的表型。在这种情况下，这将是必要的，为了证明候选人突变实际上是致病分隔其他突变有着密切的联系的利益的突变。在这种情况下，冰帽将提供一个有效的方法，打破紧紧地联系在一起的一对突变之间的联动。 </li><li> 多突变体 。它已成为常见的研究人员研究植物组合成一个单独的个人，从几个不同的位点的突变等位基因，以克服功能冗余4-6。作为一个例子，考虑一个人愿意从四个不同的位点结合成单一的植物通过基因电讯的突变等位基因的情况下。对于这个项目，将需要生成一个N个人在这四个位点的过境计划的一部分，是杂。当这个翻两番杂合子集种子，四倍纯合子预计将在目前的一率256后代造成人口。用传统的方法筛选这个难得的个人将相当劳力密集。相比之下，使用该项目的冰帽将提供一个有效的战略，确定在一个单一的一代的罕见的四倍纯合子的科学家。 </li><li> iTILLING。我们实验室最近描述了一种新的方法来筛选诱发突变基因在拟南芥中7的兴趣。此方法是在既定的技术称为园艺，用于寻找治疗与化学诱变8-10的个人订购的人口突变的变化。我们有一个名为我们的方法iTILLING，因为它是一个个性化的园艺版本，它允许一个INDividual科学家有所作为，历来需要一个大的研究小组完成。冰帽形式iTILLING方法7不可分割的一部分，如下所述。 </li></ol>背后iTILLING的想法是产生一个短暂的突变体的人口，这在传统的园艺项目创造了持久的突变人口站。 iTILLING，膨体M2的人口从种子播种到50至100冰帽块，并使用冰帽法提取DNA是，拟南芥幼苗，然后放置在4 ° C，而使用冰帽的DNA进行突变筛查PREPS。番茄幼苗被储存在12 ° C。一旦苗木携带所需的突变已经确定，他们是冰帽板回收，并转移到土壤中。 iTILLING是不打算提供一个长期持久的整个研究界的突变人口屏幕。相反，iTILLING提供了一个策略，其中一个科学家可以通过屏幕一个自定义的突变人口极少数的基因突变快速，高效，并在相对较低的成本。 我们发现冰帽是一个有效的方法，种植和基因分型的拟南芥，番茄，和水稻。我们预计，其他植​​物物种也应服从冰帽。冰帽，可容纳的各种物种的一个限制是种子的大小。只有植物种子小到足以放入一个96孔板井将在其目前的配置冰帽兼容。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> GenoGrinder </td><td> SPEX / CertiPrep，Metuchen，NJ </td><td> 2000格诺/磨床</td><td>机抖DNA提取的根板</td></tr><tr><td> ABgene热，封口机</td><td> ABgene美国</td><td> AB - 0384 </td><td>手动热合机，组织破坏前根板</td></tr><tr><td>离心机配备摆动微孔板载体</td><td> Beckman Coulter公司</td><td> Allegra的25R </td><td>任何将适当的摆动微孔板载体离心机</td></tr><tr><td> MicroFill微孔板饮水机</td><td>百特 www.biotek.com </td><td> AF1000A </td><td>自动液体灌装微孔板饮水机</td></tr><tr><td> Bialetti品牌17x11英寸的商业大片Cookie表</td><td>目标百货</td><td> www.bialetti.com </td><td>这种特定的Cookie表冰帽理想的深度</td></tr><tr><td>定制金属架</td><td>五金店</td><td></td><td>为完整描述，请参阅文本</td></tr><tr><td> 33升的塑料储物盒</td><td>目标百货</td><td> Sterilite品牌</td><td>冰帽喷泉。 塑料储物盒，大到足以容纳17x11英寸Cookie表</td></tr><tr><td> 30加仑每小时喷泉泵</td><td> Sunterra </td><td> 104506 </td><td>小型潜水喷泉泵，花园用品商店</td></tr><tr><td>喷泉泵软管</td><td>任何实验室供应公司</td><td></td><td>软管与喷泉泵兼容</td></tr><tr><td>塑料卡子</td><td>五金店</td><td></td><td>钳夹举行软管的cookie表</td></tr><tr><td>猎鹰微孔平底96孔聚苯乙烯板的透明塑料盖</td><td>屋宇署猎鹰 www.bdbiosciences.com </td><td> 351172 </td><td>透明塑料盖子覆盖冰帽育苗板</td></tr><tr><td> 3M微孔手术磁带</td><td> Fisher Scientific则</td><td> 19-027-761 </td><td></td></tr><tr><td> 3/32-inch直径不锈钢球</td><td>哈特福德技术，洛矶山，CT </td><td> 034 - 006 - 1K </td><td>收获后的根组织用来破坏</td></tr><tr><td> 3英寸的弹性发带</td><td>当地的杂货店</td><td></td><td>用于固定苗木板根板</td></tr><tr><td> 4毫米直径的木串</td><td>当地的杂货店</td><td></td><td>这些肉串通常用来准备kabobs </td></tr><tr><td>费希尔科学NUNC品牌，没有熔块1毫升的过滤板</td><td> Fisher Scientific则</td><td> 278012 </td><td> 96孔育苗盘底部的孔。 </td></tr><tr><td> Fisherbrand 96孔高大的烟囱PCR板</td><td> Fisher Scientific则</td><td> 14-230-242 </td><td> 96根板</td></tr><tr><td>琼脂 - 细胞培养试验</td><td> Sigma - Aldrich公司</td><td> A1296 </td><td></td></tr><tr><td> 4 Morpholinoethanesulfonic酸（MES） </td><td> Sigma - Aldrich公司</td><td> M2933 </td><td></td></tr><tr><td> Murashige和Skoog（MS）的基础盐的混合物</td><td> Sigma - Aldrich公司</td><td> M - 5524 </td><td></td></tr><tr><td>易撕热密封箔</td><td> ABgene美国</td><td> AB - 0745 </td><td>热封口膜密封根板前组织破坏</td></tr><tr><td>鸭品牌超宽包装胶带，3.00“X 54.6 YD </td><td> OfficeMax公司 www.officemax.com </td><td>产品编号。 21242139 </td><td>胶带密封微孔板，用于存储含有DNA的提取</td></tr><tr><td>密封辊</td><td> Bio - Rad公司</td><td> MSR - 0001 </td><td>手持到微孔板压粘接密封胶带滚动工具</td></tr><tr><td> 96孔金属热块</td><td>科尔 - 帕默</td><td> 36400-66 </td><td>用于冻结组织收获根板</td></tr><tr><td>滤纸过滤纸</td><td>瓦特曼国际</td><td> 1002 090 </td><td>用于表面消毒的种子</td></tr><tr><td>一次性巴斯德吸管</td><td> Fisher Scientific则</td><td> 13 - 678 - 6A </td><td> 5 ¾“一次性使用巴斯德吸管将种子苗木板</td></tr><tr><td>耐热烤盘（20厘米× 30厘米） </td><td>目标百货</td><td></td><td>用于构建干冰/乙醇浴。 </td></tr></tbody></table>

ice-cap: a method for growing arabidopsis and tomato plants in 96-well plates for high-throughput genotyping

它正在成为植物科学家的共同开发项目，需要大量的植物基因分型。在任何基因分型项目的第一步是从每个工厂收集组织样本。这项任务的传统方法是一个在每次一个样本植物。如果一个基因型数百或数千人的愿望，但是，使用基因分型管道中的一个重大的瓶颈这一战略的结果。冰帽，我们在这里描述的方法，高通量的解决方案，允许一个科学家收集从几千元苗在一个单一的一天1,2组织提供了这一挑战。这种级别的吞吐量是可能的，组织是从植物中收获，而不是一个- A - 96 - AT - A -，。 冰帽方法提供了一个集成平台，进行幼苗生长，组织收获，和DNA的提取。冰帽的基础是seedli增长农工商在堆放的96孔板对。上盘井含有琼脂生长介质上的个别幼苗发芽的插头。根向下生长通过琼脂培养基中，通过一个洞退出的上盘，并传递到一个较低的板块含有水。收获组织DNA的提取，在较低的板根组织水是迅速冻结，而在上盘的秧苗保持在室温。离下盘上盘，然后去皮，产生一盘96根组织冷冻样品在冰和一盘96个可行的幼苗。这项技术被命名为“冰帽”，因为它使用冰捕捉到根组织。 96孔板中，含有幼苗，然后包裹在铝箔和转移到低温。此过程中挂起的幼苗进一步增长，但并不影响他们的生存能力。一旦基因型分析已经完成，所需的基因型的幼苗可以转移到96孔p后期进一步传播的土壤。我们已经展示了使用拟南芥 ，番茄，水稻秧苗的冰帽方法的效用。我们预计，该方法也应该适用于其他物种的植物种子小到足以放入96孔板井。

Watch this Scientific Journal Video about Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping at JoVE.com

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

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

冰帽的方法使一个成长中的96孔板，每个幼苗根组织非破坏性的收获的植物。从这个根组织中提取的DNA可用于基因分型反应。我们已经发现，冰帽可以很好地用于<em&gt;拟南芥</em&gt;，番茄，水稻秧苗。

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 ...

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

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22.7K 次观看.  University of Wisconsin-Madison. 冰帽的方法使一个成长中的96孔板，每个幼苗根组织非破坏性的收获的植物。从这个根组织中提取的DNA可用于基因分型反应。我们已经发现，冰帽可以很好地用于拟南芥，番茄，水稻秧苗。

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

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

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 ...

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

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



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

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

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 ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

测量线虫寿命在96孔板

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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 ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

测量氧化应激性<em&gt;秀丽隐杆线虫</em&gt;在96孔微量滴定板

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...