我们要感谢支持沿海和公海水域低氧气地区正在进行的研究创新，不列颠哥伦比亚省知识发展基金和国家科学和工程研究理事会（NSERC），加拿大的加拿大基金会。 MT与SL支持由图拉基金会资助中心微生物的多样性和进化（CMDE）的奖学金。吨，还收到了德意志研究联合会（DFG）的奖学金支持。

Fosmid库建设已被分为四个主要步骤，并细分成几部分组成（参见图1概述） 。 步骤一（请参阅“从0.22微米Sterivex过滤器提取DNA”[3]协议） 第1部分：酶催化裂解细胞第2部分：CsCl密度梯度离心法和DNA回收净化环境的DNA 第3部分：电泳提取的DNA质量控制步骤二第1部分：恢复环境DNA酶修饰步骤环境DNA修复步骤所需的所有试剂都包含在pCC1 Fosmid图书馆震中生产套件。理想的情况下，你的基因组DNA，应调整​​为0.5μg/μL。 <ol><li>在冰上解冻的所有试剂，以下结合起来，并短暂离心​​管底部得到所有的液体： <ul><li> xμL无菌水</li><li> 8μL10X最终修复缓冲区</li><li> 8μL2.5 mM的dNTP混合液</li><li> 8μL10 mM的ATP </li><li>高达20微克剪切插入DNA（〜0.5μg/μL的） </li><li> 4μL最终修复酶的结构</li><li> 80μL的反应总体积</li></ul></li><li>在室温下孵育最小时间为45分钟到60分钟，热灭活酶组合在70 ° C为10分钟（平均时间，准备随后的最终修复大小选择一个低熔点琼脂糖凝胶DNA的，见下文）。 </li></ol>第2部分：基因组DNA的大小选择脉冲场凝胶电泳（PFGE） <ol><li>重悬在150毫升1.0X TAE 1.5克低熔点琼脂糖电泳缓冲液，并添加一个小的磁力搅拌棒。微波炉保鲜膜盖烧瓶，并迅速在微波煮沸，直到琼脂糖完全溶解。搅拌，直到它被冻倒入凝胶托盘的解决方案（〜60 ° C）。 注：不包括溴化乙锭或SYBR黄​​金在凝胶或在运行缓冲。 </li><li>组装凝胶托盘，投你的凝胶，并选择一把梳子，这将给你足够的空间来装载到一个矿两井（样缓冲液80μL，加上适当的金额）彻底结束了修复混合。 </li><li>倒入2.2 L和1.0倍TAE电泳室打开机器上的流通运行的缓冲区，并设置了冷却装置，以14 ° C泵应运行在70转，以确保足够的缓冲液的流通和缓冲区保持在14 ° C。 </li><li>倒入融化的琼脂糖凝胶托盘一个平整的表面上，一旦​​它被冷却下来，并确保您避免气泡。确保即使在融化的琼脂糖凝胶梳，否则取出梳子，放入融化的琼脂糖。凝胶固化后，取出梳子和负载的中档我PFG在第四凝胶以及标记。挤出琼脂糖凝胶注射器，切片用手术刀年底从一个小插件。以及前塞和密封与熔化的琼脂糖。放入凝胶电泳室。 注：处理您的琼脂糖凝胶非常小心低熔点凝胶制动容易。检查如果运行缓冲液降温至14 ° C，然后再开始载入您的凝胶。如果看一下您的井堵塞，用琼脂糖以下的下一次尝试：从凝胶，然后再取出梳子梳理，周围添加1-2毫升TAE缓冲，这将导致更好的井。 </li><li>其次是在第一口井负载λ的HindIII消化5μL1μLfosmid控制大小标记（震中，为100 ng /μL）。加入10μL无菌水的离心管大小的标记，以及一个样缓冲液适量。较大的体积可以更容易地加载凝胶。 fosmid标记直接加载您的最终修复和热灭活样缓冲液适量混合。盖上盖子，程序的设置。 注：我们真的建议装载不同数额的其他DNA阶梯大致量化的DNA量或可视化你的基因组DNA的剪切程度。理想的尺寸标记λDNA的HindIII精华和中档我PFG标记（自NEB）。 </li><li>程序的设置：如果您使用自动算法PFGE系统配备。该程序会自动计算出你需要单独的一个给定的尺寸范围内的DNA的设置。使用的设置是：DNA大小范围2-250 KB;校准因数：1;梯度：[6伏/厘米;运行时间：12:00时，包括角度：120 °;最初的切换时间：0.1秒，最终开关时间：21.79秒;斜坡因子a =线性。 </li><li>可视化的DNA，倒入一个容器1.0X TAE缓冲液250毫升，加入25μL的SYBR黄​​金10.000X;轻轻混匀，然后再放入您凝胶的解决方案。在黑暗中的SYBR黄​​金对光敏感1小时染色的凝胶。 注：处理您的一个garose凝胶非常小心低熔点凝胶刹车容易。 </li></ol>第3部分：DNA恢复凝胶回收DNA的提取和结扎fosmid克隆载体这部分的所有试剂，包括在震中fosmid库生产套件。 <ol><li>在平均时间，准备在水浴70℃，在45 ° C为凝胶消化步骤。 </li><li>连同一个空皮重的离心管和一个蓝色的光透射无菌手术刀把你的染色凝胶。可视化您的最终修复的基因组DNA和fosmid控制大小的标记。海关与凝胶迁移略高于fosmid控制大小标记的位置（消费这是5-7毫米宽的凝胶片）和转让的片皮重的离心管手术刀片。运行一个中档我PFG您凝胶上的标记，因为这有助于你不削减过高的凝胶。 注：之前，你把你的凝胶，以防止交叉污染和磨损观看眼镜，然后再打开蓝色的光芒，躺在蓝色的光透射保鲜膜。不要消费凝胶片，其中基因组DNA是比fosmid控制大小标记小，因为这会导致不必要的嵌合克隆。 你可以剪切和储存含有较低或较高分子量的DNA分别构建了全基因组鸟枪法或BAC文库，的凝胶片。 停止我：恢复的凝胶片（S），可在-20 ° C保存长达一年</li><li>称取皮重管和计算的凝胶片（S）的重量。琼脂糖1毫克会产生约1μL熔化琼脂糖。 </li><li>熔融低熔点琼脂糖水浴中含有你的基因组DNA在70 ° C时10-15分钟（我们不添加gelase缓冲）。凝胶片完全融化后，转让管迅速至45 ° C。 注意：不要旋涡你的样品，以帮助溶解，因为这可能会剪你的DNA。 </li><li> 1 U（加入1μl）每100μL融化的琼脂糖GELase酶制剂。在45℃孵育1小时，然后添加更多的GELase（2-4μL）孵育额外小时。热灭活的GELase酶制剂，在70 ° 10分钟。 管冰浴10分钟，离心15分钟沉淀的任何不溶性微粒的最大速度（13,000 RPM）管。小心取出上层上清转移到一个15毫升的猎鹰管和3毫升无菌水添加到管。 注意：避免任何沉淀琼脂糖吹打。当我们添加更多的GELase套件，我们为了额外GELase的酶制剂分别比。 </li><li>传输解决方案的一个Amicon Ultra - 4的离心Ultracel - 10膜过滤单元，并在4000克旋转管为6-8分钟，并丢弃的流量通过。离心机，直到溶液的过滤器上的金额减少约100〜500μL。从上面加入3毫升无菌水的另一个空猎鹰管和传输解决方案的Amicon管和重复离心步骤。第三次与3毫升水洗净，并通过再次丢弃流量。减少的最终体积为50 - 100μL。 请注意：我们使用摆动桶转子离心步骤，简单地把Amicon离心式过滤装置，在50毫升猎鹰管在离心过程中，它举行的地方。 Amicon过滤器安全地保留在过滤器上50μL的解决方案，即使延长离心。 </li><li>剩余的DNA溶液从Amicon管转移到一个预洗单片机YM - 50离心式过滤器单元和额外的50μL无菌水，收回所有DNA的Amicon管冲洗。离心机的单片机YM - 50在10,000克的微量离心过滤单元，直到过滤器仍然存在轻微的液体覆盖。如果只有少量的液体留在过滤器上，检查每分钟。打开过滤器倒置并恢复您的DNA溶液在1,000克的第二个3分钟在一个新的离心管离心步骤。由此产生的音量不得超过总数的10-15μL，否则你的DNA可能会过于稀释结扎一步。 注意：小心不要自旋过滤装置完全干燥。如果你的膜干燥，再加入10-15μl水到膜，搅拌30秒轻轻如上所述，恢复你的DNA。 </li><li>量化NanoDrop或PicoGreen检测您造成的DNA溶液。 </li><li>现在已经准备好恢复年底修复DNA的pCC1 fosmid克隆载体予以结扎。融化的冰下面的解决方案，并确保载体插入摩尔比为10:1。 注：0.5微克pCC1 - fosmid载体〜0.09 pmoles载体。 0.25微克〜40 kb的插入DNA〜0.009 pmoles插入DNA 增加在结扎步骤中使用的DNA量可能会有所帮助，如果你要获得只有少数fosmid克隆电镀后感染大肠杆菌大肠杆菌细胞包括选择板。 在下面的顺序添加试剂，并让所有液体底部短暂离心管，自来水管和旋转一次： <ul><li> xμL无菌水</li><li> 1μl的10X快速干线连接缓冲</li><li> 1μL10 mM的ATP </li><li> 1μLpCC1 fosmid载体（0.5微克/μL） </li><li> xμL集中插入DNA（0.25微克） </li><li> 1μL快速链接DNA连接酶</li><li> 10μL的反应总体积</li></ul></li><li>在室温下孵育2小时，热灭活10分钟的酶在70 ° C。 停止二：在-20 ° C存储结扎混合物或噬菌体包装的步骤进行。 注意：一个单一的连接反应将产生10 3 -10 6克隆根据环境DNA的质量。根据的估计数，特别是覆盖所需的克隆，你可以扩展连接反应。如果结扎术是扩大规模，然后噬菌体包装的提取量应扩大相应的如下所述。 </li></ol>第三步第1部分：噬菌体载体插入结扎产品包装<ol><li>打算噬菌体包装连胜前两三天内一个普通的LB平板上，在夜间在37 EPI300 - T1 ř电镀应变° C至获得单菌落。此板可密封，并保存在4 ° C以供将来使用。 </li><li>包装反应接种50 mL LB培养基中的前一天+ 10 毫米硫酸镁4（加500μL1M硫酸镁 4）在第1步中生成的板单菌落。摇在225 rpm和37 ° C过夜。 </li><li>当天的包装反应，接种5毫升在第2步准备过夜培养新鲜的50毫升LB培养基+ 10 毫米硫酸镁4。增长在37 ° C至0.8 OD 600为1.0，不超过600外径为1.0！您的样品稀释前分光光度计上测量，让你得到一个准确的结果。在冰上或储存于4℃直至进一步利用细胞。 注意：强烈建议停止三：文化可以存储长达72小时，如有必要，在4 ° C，但使用一个长大了的新鲜培养。 </li><li>冰，解冻，MaxPlax LAMBDA包装提取物，1管为每个连接反应进行了前面。解冻将需要10-15分钟。同时，1.5 mL试管置于冰上预冷。 注意：不要在冰面上留下解冻噬菌体太长。否则噬菌体感染的效率会下降。 </li><li>一旦解冻，立即转移25μl每个包装中提取的第二个预冷的1.5 mL离心管置于冰上。返回剩余的包装提取物 - 80 ° C。 注：不存储，包装用干冰或任何其他的 CO 2源提取。 </li><li>添加10μL连接反应，以每个25μL在冰上解冻提取。混合吹打几次的解决方案，避免引入气泡。短暂离心管，让所有液体的试管底部，并培育包装反应，在30 ° C为90分钟。包装提取孵化，其余的在冰上解冻后80分钟。 注意：使用30℃孵育阶段的水浴，而不是加热块。 </li><li> 90分钟包装反应完成后包装提取余下的25μL添加到每个反应管。额外的90分钟的孵育反应在30 ° C孵育90分钟后，添加100μL的准备噬菌体稀释液在每个包装管，轻轻混匀。 注意：如果您的工作效率应在年底低，你的噬菌体提取物可能是太旧或已暴露干冰或任何其他的 CO 2源。根据我们的经验，在室温30 ° C的温度，而不是最后的额外的潜伏期为2小时30（2次，每次90分钟）的噬菌体包装° C的增加导致克隆的数量。 </li><li>加入5μl氯仿每个管，轻轻地混合在一个管总体积165μL。的粘性沉淀后可能会形成氯仿此外，这不会干扰库生产。然而避免这种沉淀物以及有机氯仿相，时移液的噬菌体颗粒。 停止四：在这一点上包装的噬菌体颗粒，可用于储存数天在​​4 ° C 注：最好的噬菌体感染效率，使用新鲜包装的噬菌体。 </li></ol>第2部分：库主机大肠杆菌感染大肠杆菌 <ol><li>添加包装噬菌体EPI300 - T1 ř宿主细胞（OD 600 = 0.8-1.0），在400μLEPI300 - T1 R为每10μL噬菌体颗粒细胞的比例。为了避免吸取我们使用任何氯仿125μL打包的噬菌体颗粒和5毫升准备EPI300 - T1 ř宿主细胞。轻轻混匀，然后在37 ° 30分钟的彗星。 注意：要小心不转让氯仿的宿主细胞，当您从噬菌体包装管的噬菌体颗粒。如果你不确定，采取打包的噬菌体颗粒。 </li></ol>第3部分：电镀和滴定<ol><li>四个小LB + 12.5微克/ mL氯霉素的培养皿中添加5-7灭菌的玻璃珠。 </li><li> 50μl和两次10μL噬菌体感染了四个独立的板块EPI300 - T1 R细胞的两次。为了确保甚至蔓延，添加一些LB肉汤（〜50μL）板块与10μL噬菌体感染细胞。摇板水平均匀分布在板的细胞。让板干燥15分钟，然后取出反相板的珠子。 注：这些板块是用来确定转化的数量，并计算在殖民地采摘一步需要适当稀释，以避免过高的殖民地密度。 </li><li>将板倒挂在37℃培养箱中培养16至24小时至48小时，直到殖民地形式。 24小时后，检查板，防止快速增长克隆生长到邻居殖民地。 </li><li>同时，收获从5毫升感染，在3,500克10分钟，在4 ° C离心遗留的残余细胞上清液。管中添加1毫升LB / 20％的甘油。重悬细胞沉淀和分装到100μL各2毫升离心管管。立即冻结，并储存在-80 ° C为进一步利用采摘一步殖民地。 </li><li>第二天，计数克隆板的数量，并确定滴度。确保包括正确的稀释倍数来计算总额fosmid克隆。 注意：此过程会产生在10,000和50,000总fosmid克隆之间;然而根据不同的环境中提取的DNA的质量和纯度，观察范围之间3000高达80,000 fosmid克隆。 </li></ol>第四步第1部分：琼脂和孔板准备<ol><li>倒入盘如下媒体;包括12.5微克每毫升LB培养基，氯霉素： <ul><li> 100X15mm培养皿：25毫升LB琼脂</li><li> 150X15mm培养皿：50ML LB琼脂</li><li> 245X245mm平方米的生物测定板：250毫升LB琼脂</li></ul>注：重要的是要倒一个平整的表面上的媒体，使整个板块的深度均匀琼脂。我们建议使用245X245毫米板fosmid库，这样你就不会必须处理许多板。 </li><li>解冻甘油含冰准备fosmid克隆。扩散板，你要使用的基础上计算滴度适当的金额（编制甘油此外集中你的细胞〜5倍）。细胞生长的孵化时间为24小时，在37 ° C。 停止五：当殖民地的形成，存储长达一个月的密封的琼脂平板上，在4 ° C，直到他们在殖民地采摘一步。 注：殖民地需要均匀分散，相互遥远足够的，应该生长在直径约1毫米。使用玻璃珠电镀细胞能产生一个殖民地之间的良好分离。在一个100X15mm培养皿中，通常150〜250菌落是最高的机器人采摘效率和良好的密度为22 × 22厘米，不超过2,000个殖民地获得。 </li><li>在殖民地采摘时代，准备96或384孔板充满12.5微克每毫升肉汤氯霉素，以及20％的甘油库存储在-80为辅的LB肉汤° C。孔板充满QFill3板自动灌装系统，如下所述。 </li><li>高压灭菌器2台（500毫升）玻璃瓶，盖子和硅胶管。不要没有反应釜多方面的，包括针组件和安装法兰。组装QFill3靠近本生灯火焰或引擎盖内创造和保持无菌环境。 注意：确保按捏阀激活完全进入阀和滑动油管，硅胶管插入到夹管阀。 （制动像配药中期到下一列本工程）。 </li><li>消毒一次通过〜50毫升1％的漂白粉蒸压生署2，以及80％的乙醇，一个传递点胶针头和收集废物的解决方案的平台上放置一个提示框盖。灭菌后，更改其他设置，瓶，盖和硅管组成。 </li><li>填写〜300毫升培养基瓶。运行足够的媒介，通过以摆脱任何剩余的乙醇清洗步骤。一旦油管清洗，加载384分之96以及板的平台上。按“开始”，填补你的盘子，每孔应与200μLLB肉汤准备填补。要干净点胶针，运行〜80％的乙醇50毫升。 注意：确保您的孔板是在灌装前的标记。 </li></ol>第2部分：殖民地采摘机器人设置<ol><li>我们使用的是克隆采摘机器人QPix2 AC盘带至用户手册。用户应该熟悉处理他们的机器人，以及如何建立一切以保证最高的采摘效率。 </li><li>克隆采摘区周围建立一个无菌的环境，开启紫外灯30分钟。虽然在紫外线照射下，准备500毫升1％的漂白粉，500毫升无菌生署2 O和500毫升80％乙醇。当紫外线光切断托盘，填补了标记。始终倒入标示正确的解决方案托盘的解决方案。从后到前填写1％漂白粉，然后高压灭菌的dh 2 O和80％的乙醇在前面托盘。确保他们是全职。采摘针不能没有足够的洗涤液洗。 注：显示器80％的乙醇，随着时间的推移。它会蒸发，需要突破。 </li><li>设立最佳采摘参数后，将准备好的琼脂平板fosmid克隆和孔板之间的Q - PIX工作区的职位。确保你已经采取所有的板块涵盖关闭！ </li><li>板应该对右前方角落紧，所以它不能移动时采摘。然后关闭前面板。 注：这是非常重要的的！ </li><li>一旦所有的参数设置就可以开始采摘过程。当完成所有采摘冲洗与去离子水，并设置画笔和托盘在板凳上干。清理任何可能发生的泄漏，并用80％乙醇擦拭里面的Q - PIX。 </li></ol>第3部分：孵育过夜和库存储<ol><li>确保所有甘油股票板块标记，并将其放置在37℃培养箱中培养24小时。离开孵化器的说明，包含日期，培养板的数量，培养时间，搬迁时间和您的姓名缩写。 </li><li>对于存放时间过长，放入一个-80 ° C冰箱过夜孵育。 </li></ol>代表性的成果： 协议描述的程序如何生成环境fosmid库捕捉到一个给定的栖息地中的微生物群落的遗传内容。这个协议应该创建一个fosmid库，是有代表性的的采样环境的遗传内容，并应使读者能够修改和优化的关键步骤，如果在整个过程结束时得到的fosmid克隆的数额太低。

<ol>
	<li>Zaikova, E., Hawley, A., Walsh, D. A., Hallam, 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=Seawater+sampling+and.">Seawater sampling and.</a> J Vis Exp. , (2009).</li><li>Walsh, D. A., Zaikova, E., Hallam, 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=Large+volume+(20L+)+filtration+of+coastal+seawater+samples.">Large volume (20L+) filtration of coastal seawater samples.</a> J Vis Exp. 28, (2009).</li><li>Wright, J. J., Lee, S., Zaikova, E., Walsh, D. A., Hallam, 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=DNA+Extraction+from+0.22+&#956;M+Sterivex+Filters+and+Cesium+Chloride+Density+Gradient+Centrifugation.">DNA Extraction from 0.22 &#956;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation.</a> J Vis Exp. 31, 10-3791 (2009).</li><li>Stein, J. L., Marsh, T. L., Wu, K. Y., Shizuya, H., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+uncultivated+prokaryotes:+isolation+and+analysis+of+a+40-kilobase-pair+genome+fragment+from+a+planktonic+marine+archaeon.">Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.</a> J. Bacteriol. 178, 591-599 (1996).</li><li>B&#233;j&#224;, O., Suzuki, O., Koonin, M. T., Aravind, E. V., Hadd, L., Nguyen, L. P. A., Villacorta, R., Amjadi, M., Garrigues, C., Jovanovich, S. B., Feldman, R. A., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+analysis+of+bacterial+artificial+chromosome+libraries+from+a+marine+microbial+assemblage.">Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage.</a> Environ. Microbiol. 2, 516-529 (2000).</li><li>DeLong, E. F., Preston, C. M., Mincer, T., Rich, V., Hallam, S. J., Frigaard, N. -. U., Martinez, A., Sullivan, M. B., Edwards, R., Brito, B. R., Chisholm, S. W., Karl, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community+genomics+among+stratified+microbial+assemblages+in+the+Ocean's+interior.">Community genomics among stratified microbial assemblages in the Ocean's interior.</a> Science. 311, 496-503 (2006).</li><li>Hallam, S. J., Putnam, N., Preston, C. M., Detter, J. C., Rokhsar, D., Richardson, P. M., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+methanogenesis:+testing+the+hypothesis+with+environmental+genomics.">Reverse methanogenesis: testing the hypothesis with environmental genomics.</a> Science. 305, 1457-1462 (2004).</li></ol>

一个过程描述了如何最有效地产生大插入fosmid从沿海水样的基因组DNA库。上游基因组DNA的提取是在一个单独的协议[3]。 由于fosmid库生产是一个多步骤的过程中，计划整个过程，包括提出的所有四个步骤的时间至少需要两到三个星期。基因组DNA的提取是最关键的一步，所有下游步骤依靠提取的基因组DNA的质量和数量;，所以考虑这一步花费足够的时间和工作细心。您提取的DNA的?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Step II</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CopyControl&trade; Fosmid Library Production Kit</td>
<td>&nbsp;</td>
<td>EPICENTRE</td>
<td>CCFOS110</td>
<td>sufficient to generate 9-10 fosmid libraries</td>
</tr>
<tr>
<td>SeaPlaque Agarose</td>
<td>&nbsp;</td>
<td>Cambrex</td>
<td>50100</td>
<td>&nbsp;</td>
<tr>
<td>stirring hot plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>PC-420D</td>
<td>&nbsp;</td>
<tr>
<td>1.0X TAE running buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CHEF Mapper XA pulsed field electrophoresis system including cooling module and variable speed pump</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>&lambda; DNA-HindIII Digest</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>N3012S</td>
<td>&nbsp;</td>
<tr>
<td>MidRange I PFG Marker</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>N3551S</td>
<td>&nbsp;</td>
<tr>
<td>10,000X SYBR Gold</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S11494</td>
<td>&nbsp;</td>
<tr>
<td>microwavable plastic wrap</td>
<td>&nbsp;</td>
<td>Saran premium wrap</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Safe Imager transilluminator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S37102</td>
<td>&nbsp;</td>
<tr>
<td>GELase Agarose Gel-Digesting Preparation</td>
<td>&nbsp;</td>
<td>EPICENTRE</td>
<td>G09100</td>
<td>&nbsp;</td>
<tr>
<td>scalpel</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-927-5D</td>
<td>&nbsp;</td>
<tr>
<td>Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC801024</td>
<td>&nbsp;</td>
<tr>
<td>Microcon YM-30 Centrifugal Filter Unit </td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>42410</td>
<td>&nbsp;</td>
<tr>
<td>nuclease free water</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM9932</td>
<td>&nbsp;</td>
<tr>
<td>Quant-iT PicoGreen dsDNA Assay Kit *2000 assays*#</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P7589</td>
<td>&nbsp;</td>
<tr>
<td>digital dry block heater</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>12621-088</td>
<td>&nbsp;</td>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14231-854</td>
<td>&nbsp;</td>
<tr>
<td>centrifuge</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>Avanti J-E</td>
<td>&nbsp;</td>
<tr>
<td>centrifuge rotor</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>JA 5.3</td>
<td>&nbsp;</td>
<tr>
<td>table top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5415D</td>
<td>&nbsp;</td>
<tr>
<td>microcentrifuge tubes, 1.7 mL, clear</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>MCT-175-C</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes </td>
<td>&nbsp;</td>
<td>Fisher-Corning</td>
<td>430052</td>
<td>&nbsp;</td>
<tr>
<td>Step III</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LB broth</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP 1426-2</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M2643</td>
<td>&nbsp;</td>
<tr>
<td>phage dilution buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM Tris-HCl [pH 8.3], 100 mM NaCl, 10 mM MgCl2</td>
</tr>
<tr>
<td>cryogenic vials</td>
<td>&nbsp;</td>
<td>Fisher-Corning</td>
<td>430488</td>
<td>&nbsp;</td>
<tr>
<td>Step IV</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>96 well plate with lid</td>
<td>&nbsp;</td>
<td>Fisher-Corning</td>
<td>CS003370(3370)</td>
<td>&nbsp;</td>
<tr>
<td>384 well plate with lid</td>
<td>&nbsp;</td>
<td>Fisher-Corning</td>
<td>07201157(3680)</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes 100 O.D. x 15mm H</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes 150 Dia. x 15mmH</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-757-14</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon BioDish XL 245 X 245 mm</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CABD351040</td>
<td>&nbsp;</td>
<tr>
<td>glycerol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G5516</td>
<td>&nbsp;</td>
<tr>
<td>chloramphenicol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C0378</td>
<td>&nbsp;</td>
<tr>
<td>LB broth</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP 1426-2</td>
<td>&nbsp;</td>
<tr>
<td>Difco Agar</td>
<td>&nbsp;</td>
<td>BD</td>
<td>214530</td>
<td>&nbsp;</td>
<tr>
<td>glass beads</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>10-310-1</td>
<td>&nbsp;</td>
<tr>
<td>QFill3</td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>X3050</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>Javex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E7023</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>Fisher-Corning</td>
<td>430052</td>
<td>&nbsp;</td>
<tr>
<td>QPix colony picker</td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>QPix2</td>
<td>&nbsp;</td>
<tr>
<td>picking head (E. coli) </td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>X4006</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

large insert environmental genomic library production

微生物在自然界中的绝大多数，目前仍无法进入传统的种植方法。在过去的十年中，独立的文化环境基因组（<em&gt;即</em&gt;宏基因组）的方法已经出现，使研究人员的桥梁，通过直接从环境中捕获的土著微生物群落的遗传内容种植的差距。为此，尽管巧妙的实验室克隆技术，基因组DNA库使用标准。在这里，我们描述了与经季节性缺氧峡湾垂直深度连续的DNA插入一个大环境fosmid基因库的建设。该协议是直接挂钩的一系列连接的协议，包括沿海海水取样[1]，微生物生物量大量过滤[2]和DNA提取和纯化协议[3]。首先，高品质的基因组DNA与创造的5 - 磷酸两端钝月底修复。最终修复DNA的脉冲场凝胶电泳（PFGE）的大小选择和执行收回30至60万个碱基对的长度（KB）的DNA片段凝胶萃取。大小选择的DNA纯化远离PFGE凝胶基质和结扎磷酸酶治疗钝fosmid CopyControl载体pCC1（震中<a href="http://www.epibio.com/item.asp?ID=385"&gt; http://www.epibio.com/item.asp?ID=385</a&gt;）。 pCC1和插入DNA的线性concatemers随后headfull的lambda terminase包装成噬菌体颗粒，随后感染的抗噬菌体<em&gt; E。大肠杆菌</em&gt;细胞。抗生素的选择下，成功转染克隆LB琼脂平板上恢复和384孔板使用一个自动化的殖民地采摘机器人（Qpix2，GENETIX）的格式存档。目前的协议，吸引了来自各种来源，包括CopyControl Fosmid图书馆震中生产套件多个研究小组已经发表的作品[4-7]。每一步都是与心目中的最佳做法。只要有可能，我们强调在执行细微之处，以提高整体质量和图书馆的生产效率。 fosmid库生产和自动化殖民地采摘的整个过程至少需要7-10天，因为有很多孵化步骤包括。然而，有几个停车点可能是在协议中提到的。

Watch this Scientific Journal Video about Large Insert Environmental Genomic Library Production at JoVE.com

Large Insert Environmental Genomic Library Production

是描述一个与环境基因组DNA经季节性缺氧峡湾的垂直深度连续分离的fosmid库建设。由此产生的克隆库回升到384孔板和存档，为下游的测序和一个自动化殖民地采摘系统的应用功能筛选。

大型插入环境基因组文库生产

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

large-insert-environmental-genomic-library-production

Research

JoVE Journal

Biology

15.8K 次观看.  University of British Columbia - UBC. 是描述一个与环境基因组DNA经季节性缺氧峡湾的垂直深度连续分离的fosmid库建设。由此产生的克隆库回升到384孔板和存档，为下游的测序和一个自动化殖民地采摘系统的应用功能筛选。

视频: Large Insert Environmental Genomic Library Production

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.

宏基因组库的大型屏幕

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

科研

生物学

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

全基因组阵列比较基因组杂交技术示范

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

为贴壁细胞培养的氧气插入的制备及操作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

基因改造的Picoeukaryote<em&gt; Ostreococcus金牛</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

阵列比较基因组杂交（CGH阵列），用于检测基因拷贝数变异的

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.