安捷伦Stratagene公司产品部 <ul><li> Harini拉维</li><li>纳塔利娅Novorodovskaya </li><li>斯科特Basehore </li><li>杰夫Braman </li><li>雷切尔福尔摩沙</li><li> Ronda的艾伦</li><li>拉杰什Bagga </li><li>德里克厅</li><li>莎拉Jandle </li><li>丹尼尔霍夫曼</li></ul> 安捷伦实验室 <ul><li>罗伯特金凯德</li><li>玛丽麦克布赖德</li></ul> 安捷伦欧洲 <ul><li>奈杰尔斯金纳</li><li>尤尔根施奈德</li><li>斯蒂芬穆勒</li><li>马丁Kratzmeier </li></ul> 坎普登BRI <ul><li>史蒂夫加勒特</li></ul>

1：准备鱼样本DNA提取<ol><li>每条鱼样本进行测试，将一块鱼组织，体重10毫克，1克（生或熟）之间，成一个单一的1.5 ml的离心管。使用下面的数字估算基于样本大小的生鱼片样品的重量的方针。 <img src="/files/ftp_upload/1871/1871fig1.jpg" alt="图1" /> 图1。基于对样本大小的鱼类样品的质量评估。 </li><li>预温蛋白酶K消化缓冲至65℃，在孵化器或水浴5分钟。 </li><li>准备工作液200μL蛋白酶K消化缓冲液和20μL蛋白酶K，每样品相结合的蛋白酶K。 注意：每次使用前准备工作的一个新的蛋白酶K的解决方案 。 </li><li>加入220μl蛋白酶K工作液每个鱼样品1.5 ml管的。孵育管在65℃的孵化器或水浴10分钟。 </li><li>自旋为3 5 XG颗粒任何未消化的组织，在14000分钟的离心管。 </li><li>转移到一个新的1.5 ml管每个上清液150μL。上清这些管子管或任何油性物质，可目前在管的顶部底部，避免传输任何未消化的物质。样品从它的基因组将提取的DNA。 </li></ol> 2：提取基因组DNA <ol><li>在无菌容器（聚丙烯管或玻璃瓶），编制了环丁砜和核酸结合缓冲液中的工作液320μl的90％，环丁砜和每个样品核酸结合缓冲液170μL结合。 您可能要准备一个足够数量的这种混合过程，还有很多的样品，为你计划，以审查在未来4个星期。 这种混合物可以存储在室温下长达30天。避免暴露在储存过程中的混合物光。 </li><li>加入490μL的环丁砜/结合缓冲液的混合物（在上述步骤1准备）每条鱼样本。涡或吸管直到均质样品反复。这种混合物的加入将使每个样品的总体积为640μL。 </li><li>将每个样品2毫升插座管内已就位（已提供），并捕捉到的旋杯的顶部管第一个DNA结合自旋杯。 </li><li>自旋为1分钟，在14000 XG微量的样品加载到自旋杯矩阵的DNA。 </li><li>删除和保留的旋转杯和丢弃滤液。对于每个样本，更换插座中的管旋杯1X高盐的洗涤缓冲液，然后加入500μl和盖管。 </li><li>在1分钟14,000 XG离心旋转样品。 </li><li>删除和保留的旋转杯和丢弃滤液。对于每个样本，更换插座中的管旋杯，再加入80％乙醇500μl和盖管。 </li><li>在1分钟14,000 XG离心旋转样品。 </li><li>重复步骤7和8两个更次，共3洗用500μL80％乙醇。 </li><li>在80％乙醇的第三洗后，删除和保留的旋转杯和丢弃滤液。更换插座管旋杯和14000 XG自旋为2分钟离心干燥纤维基质。 </li><li>将1.5 - M1收集管旋杯。直接在杯子里面的纤维基质，加入100μL洗脱缓冲液每次旋转杯。卡上旋杯收集管帽，并在室温下孵育1分钟。 </li><li>在以最大速度离心1分钟的旋转样品。 </li><li>纯化的DNA洗脱缓冲液的离心管。丢弃旋转杯和盖管。在4 ° C的DNA可以存储长达一个月。对于长期储存，储存的DNA在20 ° C或80 ° C。 </li><li>如果需要的话，你可以在分光光度计测量浓度的DNA样本。 </li></ol> 基因组DNA提取协议通常收益率与浓度范围从5 ng /μL的500 ng /μL的样品。 的PCR - RFLP协议的工作原理与DNA不等从0.05 ng /μL的2000毫微克/μL的样品井。 3：设置PCR反应<ol><li>准备用32μL无菌，无DNase的水相结合，8μL的DNA股票的积极控制鲑鱼DNA 50 ng /μL的稀释。涡旋混合。 稀释后的样品可在4 ° C储存供日后使用。 </li><li>准备组件相结合，在下面，以表的反应。所有将要运行的同时，通过扩大中列出的卷的PCR反应准备一个单一的试剂混合表。此外测试的DNA样本，包括阳性对照反应和无模板的对照反应。建议准备重复每个测试的DNA样本的PCR反应。准备好你的反应，加上一个反应体积超过足够的试剂混合。 例如，如果你有5个测试的DNA样本， 准备为8的反应（5测试反应，1个阳性对照，无模板控制， 和 1过剩）或13反应包括足够的试剂混合，如果你的测试反应的重复（10重复试验的反应，1个阳性对照，1无模板控制和1过剩） 。 PCR试 ​​剂混合 <table border="1"><tbody><tr><td> 组件 </td><td> 卷1反应 </td><td> 体积5的反应 </td></tr><tr><td> DH 2 O，无菌</td><td> 9μL </td><td> 45μL </td></tr><tr><td> 2X PCR扩增预混</td><td> 12.5μL </td><td> 62.5μL </td></tr><tr><td>引物混合</td><td> 2.5μL </td><td> 12.5μL </td></tr><tr><td>总成交量</td><td> 24μL </td><td> 120μL </td></tr></tbody></table></li><li>涡试剂混合，然后分发到每一个人的薄壁PCR反应管24μL。 </li><li>加入1μL稀释的阳性对照DNA阳性对照反应管。为了测试样品管，加入1μL测试的DNA样本。对于没有模板控制反应，加入1μLDNase的自由水的地方的DNA。 为了避免交叉污染，使用一个新鲜的吸管尖，每个DNA样本。 加入样品后，迅速 ​​移液管的内容向上和向下混合反应。 </li><li>第反应管，涡旋管简单地混合和离心管。 </li></ol> 4：运行PCR议定书“ <ol><li>在热循环仪的反应和运行PCR程序如下所示。 PCR循环协议 <table border="1"><tbody><tr><td> 分部 </td><td> 循环数 </td><td> 温度 </td><td> 时间 </td></tr><tr><td> 1 </td><td> 1 </td><td> 95℃ </td><td> 5分钟</td></tr><tr><td> 2 </td><td> 40 </td><td> 95℃ 50℃ 72℃ </td><td> 30秒 30秒 30秒</td></tr><tr><td> 3 </td><td> 1 </td><td> 72℃ </td><td> 7分钟</td></tr></tbody></table></li></ol> 5：精华PCR产物用限制性内切酶<ol><li>标签0.5毫升管或0.2毫升地带管，使用限制性内切酶消化反应。每个PCR反应将被消化了三种不同的限制性内切酶DdeI，HaeIII和NlaIII。因此，每个PCR反应，PCR样品的名称和限制性内切酶的名称与标签的三个独立的管。 </li><li>以下组件相结合，以准备DdeI消化试剂混合。准备所有DdeI消化反应，利用每个组件的倍数（另加至少有一个反应体积过量）单一试剂的混合物。 PCR完成后，PCR反应限制性内切酶限制性片段长度多态性（RFLP）分析处理。 DdeI消化试剂混合 <table border="1"><tbody><tr><td> 组件 </td><td> 卷 </td></tr><tr><td> DH 2 O，无菌</td><td> 1.5μL </td></tr><tr><td> 10X DdeI缓冲区</td><td> 0.5μL </td></tr><tr><td> 10X DdeI酶</td><td> 0.5μL </td></tr></tbody></table></li><li>涡试剂混合，然后分发到个人的反应，为DdeI标记的试管2.5μL。 </li><li>以下组件相结合，以准备HaeIII消化试剂混合。准备所有HaeIII消化反应，利用每个组件的倍数（另加至少有一个反应体积过量）单一试剂的混合物。 HaeIII消化试剂混合 <table border="1"><tbody><tr><td> 组件 </td><td> 卷 </td></tr><tr><td> DH 2 O，无菌</td><td> 1.5μL </td></tr><tr><td> 10X HaeIII缓冲区</td><td> 0.5μL </td></tr><tr><td> 10X HaeIII酶</td><td> 0.5μL </td></tr></tbody></table></li><li>涡试剂混合，然后分发到个人的反应，为HaeIII标记的试管2.5μL。 </li><li>准备试剂混合NlaIII消化相结合，以下面的组件。准备为所有NlaIII消化反应，利用每个组件的倍数（另加至少有一个反应体积过量）的单一试剂的混合物。 NlaIII消化试剂混合 <table border="1"><tbody><tr><td> 组件 </td><td> 卷 </td></tr><tr><td> DH 2 O，无菌</td><td> 1.5μL </td></tr><tr><td> 10X NlaIII缓冲区</td><td> 0.5μL </td></tr><tr><td> 10X NlaIII酶</td><td> 0.5μL </td></tr></tbody></table></li><li>涡试剂混合，然后分发到个人的反应，为NlaIII标记的试管2.5μL。 </li><li>对于每个消化反应，添加适当的PCR产物2.5μL标记的试管。测试PCR反应以及作为阳性对照反应都需要与所有三个限制性内切酶消化。 </li><li>涡的消化反应，然后短暂离心管。 </li><li>孵育所有在37 ° C 2小时的消化反应。此潜伏期可进行热循环。如果需要的话，反应可能会留在37 ° C过夜。 </li><li>转移至65 ° C 15分钟的反应。这一步可以在热循环仪。 </li><li>添加1μL60 mM的EDTA（套件），以及每个反应和涡。 </li></ol> 6：分析限制性内切酶消化模式<ol><li>准备凝胶染料混合，放置在芯片上的吸站的DNA芯片，并加载的DNA芯片上的混合。 </li><li>吸取5μLDNA标记与梯形图符号和标记到每个在芯片上的12个样品孔。 DNA分子标记在DNA 1000试剂盒在绿色覆盖的管。 </li><li>吸取1μLDNA梯状到梯子的象征标记。 DNA梯状提供了一个黄色的雪山管的DNA 1000试剂盒。 </li><li>对于每一个DNA样本，吸管1μL酶切反应每个12芯片的样品孔，根据下图中的的指导方针之一。使用这种方法，3反应为阳性对照样品的消化，井1-3 4-6井的3个试验样品1，井7-9消化反应为3个测试样本2的消化反应， 10-12井3.Analyze安捷伦2100生物分析仪上的酶切反应测试样品，以确定在消化过程中产生的片段长度为3消化反应。安捷伦DNA 1000工具包指南上运行的生物分析实验室芯片的完整说明。为方便起见，下面提供了一个简短的协议。 <img src="/files/ftp_upload/1871/1871fig2.jpg" alt="图2" /> 图2。组织芯片的样品。 如果您有未使用的水井不到4个DNA样本，吸管1μL水 。 如果你有超过4个DNA样本，你将需要运行多个芯片 。 请注意，它是没有必要的每一块芯片上运行的阳性对照样品。 </li><li>水平放置的芯片，在2400转的德国IKA旋涡混合器和60秒的旋涡适配器。 </li><li>安捷伦2100生物分析仪芯片插入并启动芯片运行。传入的原始信号显示在仪器中。 运行完成后，峰被确定为所有使用的高峰期的设置，发现算法的样本。 如果您认为该算法无法检测到真正的高峰，你可能会降低高度阈值的设定值，允许该算法确定高峰。 </li><li>转到的含量范围内，选择芯片的摘要“选项卡。在“样品名称”字段中，输入一个如下图所示的12口井在芯片上的所有样品名称。 <img src="/files/ftp_upload/1871/1871fig3.jpg" alt="图3" /> 图3。摘要标签芯片的样品名称条目。 </li></ol> 7：确定使用RFLP分析比对测试样品的物种安捷伦软件应用RFLP解码器可用于为基于在消化反应所产生的片段长度的DNA样本测试，以确定鱼的种类。表7阳性对照限制性内切酶预期的产品尺寸117（BP）DdeI DNA片段大小340，332，459 NlaIII HaeIII 40，105，333，这里提供的指示RFLP匹配器使用的默认分析设置。请参考软件的帮助系统上运行软件和解释显示的详细信息。 <ol><li>启动RFLP解码程序。 </li><li>点击文件&gt;打开&gt; XAD文件。 打开对话框WILL打开。 </li><li>选择XAD文件，其中包括酶切反应的DNA芯片。点击“ 打开 ”。 将打开一个对话框，列出了在芯片上加载的样品。 </li><li>每个样品用酶 “栏下的下拉菜单选择相应的一个DNA样本的三个消化反应，并指定相应的限制性内切酶。在下图中，已填写的酶列的DNA样本1。 <img src="/files/ftp_upload/1871/1871fig4.jpg" alt="图4" /> 图4。指定对每一个样品的限制性内切酶。 </li><li>标记为较低的标记“％分钟的峰高的领域，默认值是10.0％，如果需要的话，这可能会降低价值，以确定被错过的小峰，或提出放弃在从非特定的噪音造成的峰电泳。点击作出任何调整，以最小的峰高值后，重新融入社会。 <img src="/files/ftp_upload/1871/1871fig5.jpg" alt="图5" /> 图5。分配的最小峰值高度。 </li><li>单击对话框底部的计算器 。 片段长度从生物分析仪运行获得的数据将填充的软件领域。 </li><li>在比分在屏幕上方左上角的下拉列表，选择合适的算法对数据的分析。如果由一个单一的鱼种鱼样品正在分析中，选择在下拉列表中的得分骰子（李内）。如果样本包括物种的混合物中， 选择 “混合“。 在屏幕底部的标记的综合评分表列出了最好的品种匹配所有三个消化反应的结果为基础。表中列出的物种名称，以及共同的名字（见下文）。以绿色突出显示的完美匹配（1分）。以黄色突出显示的比赛被认为是附近的匹配。您可以双击一个物种的名称，使该物种的FishBase网页。 <img src="/files/ftp_upload/1871/1871fig6.jpg" alt="图6" /> 图6。基于对消化物种鉴定。 </li><li>在较低的截止和匹配公差域在屏幕的上方，你可以调整默认设置，以改善物种的最佳匹配得分。 较低的临界值是用来丢弃任何比在现场指定的长度较短的片段。本场比赛的公差值决定如何关闭一个片段长度必须被认为是匹配的预测片段。 </li><li>其余的，这同一个芯片上的DNA样本4重复步骤8。 </li></ol> 8：代表性的成果： 一个Bioanlyzer凝胶图像与来自4个不同的鱼种的限制性消化样品如下图所示。预期结果为阳性的DNA样本，总结于下表。 <img src="/files/ftp_upload/1871/1871fig7.jpg" alt="图7" /> 图7。限制性内切酶消化反应样品的生物分析仪凝胶图像。每个凝胶泳道是在这一反应中所用的限制性内切酶标记。 三文鱼阳性对照预期的DNA片段大小<img src="/files/ftp_upload/1871/1871fig8.jpg" alt="图8" /> 图8。三文鱼阳性对照预期的DNA片段大小。

<ol>
	<li>Dooley, J. J., Sage, H. D., Brown, H. M., Garrett, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+fish+species+identification+by+use+of+lab-on-a-chip+technology.">Improved fish species identification by use of lab-on-a-chip technology.</a> Food Control. 16 (7), 601-607 (2004).</li><li>Dooley, J. J., Sage, H. D., Clarke, M. -. A. L., Brown, H. M., Garrett, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+species+Identification+Using+PCR-RFLP+Analysis+and+Lab-on-a-Chip+Capillary+Electrophoresis:+Application+to+Detect+White+Fish+Species+in+Food+Products+and+an+Interlaboratory+Study.">Fish species Identification Using PCR-RFLP Analysis and Lab-on-a-Chip Capillary Electrophoresis: Application to Detect White Fish Species in Food Products and an Interlaboratory Study.</a> J. Agric. Food Chem. 53, 3348-3357 (2005).</li><li>Dooley, J., Garrett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+PCR-RFLP+Profiles+for+Fish+Species+Using+the+Agilent+2100+Bioanalyzer.">Determination of PCR-RFLP Profiles for Fish Species Using the Agilent 2100 Bioanalyzer.</a> Agilent Technologies Application Note. , (2005).</li><li>Dooley, J. J., Clarke, M. L., Sage, H. D., Brown, H. M., Garrett, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+chip-based+capillary+electrophoresis+system+to+enable+simple+PCR-RFLP+identification+of+fish+species.">Application of a chip-based capillary electrophoresis system to enable simple PCR-RFLP identification of fish species.</a> FSA Final Report Q01069. , (2004).</li></ol>

作者受雇于产生本文中使用的试剂和仪器的安捷伦科技。

我们展示一个海鲜产品中的鱼类物种的识别简单，快速，准确的DNA为基础的筛查方法。这强大的方法提供了在不到一个工作日的重复性好，精确的结果，它可以在商业测试设施，由于简化的协议和进行简单的设置实现。它的特点是客观数据分析多态性解码器软件使用实验衍生的鱼类型材的可扩展数据库的生成。 这种测试方法与常规的实施，有潜力，以防止错误标签的海鲜产品，并协助在保持合适的准确记录与政府法规。

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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>Agilent 2100 Bioanalyzer (with chip priming station and IKA vortex mixer)</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2939AA</td>
<td>&nbsp;</td>
<tr>
<td>Agilent 2100 Bioanalyzer laptop &amp; Expert Software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2953CA</td>
<td>G2950CA - desktop PC &amp; Expert Software</td>
</tr>
<tr>
<td>Agilent Bioanalyzer DNA 1000 Reagent Kit (part #5067-1504)</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>5067-1504</td>
<td>&nbsp;</td>
<tr>
<td>Thermal Cycler</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>PCR Strip Tubes</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>401428</td>
<td>&nbsp;</td>
<tr>
<td>PCR Strip Caps</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>401425</td>
<td>&nbsp;</td>
<tr>
<td>96-Well Working Rack</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>410094</td>
<td>&nbsp;</td>
<tr>
<td>Fish Species Identification System DNA Isolation Kit</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>Contact</td>
<td>&nbsp;</td>
<tr>
<td>Fish Species Identification System PCR-RFLP Reagent Kit</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>Contact</td>
<td>&nbsp;</td>
<tr>
<td>RFLP Matcher Software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>Contact</td>
<td>&nbsp;</td>
<tr>
<td>100% ethanol, 200 proof (USP grade or equivalent)</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Sterile, nuclease-free water</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Pipettors</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Incubator or water bath set to 65&deg;C</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Sterile glass bottle or polypropylene tube (e.g. 14-ml BD Falcon polypropylene)</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>round-bottom tubes or 50-ml BD Falcon polypropylene conical tubes)</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge tubes 1.5ml</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Tube rack 1.5ml</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Pipette Tips</td>
<td>&nbsp;</td>
<td>Various</td>
<td>Various</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

dna-based fish species identification protocol

我们已经开发出一种快速，简便，准确的DNA为基础的筛查方法来识别存在于新鲜和加工的海产样本的鱼类物种。这种多用途的方法采用PCR扩增从鱼样本中提取的基因组DNA，通过限制性片段长度多态性（RFLP）分析生成的片段，可以解决在Agilent 2100生物分析仪及配套使用RFLP图谱匹配软件的正确物种的模式。 鱼的鉴别方法使用简单，可靠，自旋基于列的协议，从鱼样本中分离出DNA。用蛋白酶K释放到溶液中的核酸样品的处理。然后分离出的DNA结合缓冲液中的悬浮样品和装载含有硅为基础的光纤矩阵的杯子到微观自旋。样品中的核酸结合到纤维基质。固定的核酸洗涤以去除污染物，并在终体积为100μL收回总DNA。分离出DNA的发现在所有的鱼类基因组的序列绑定到所提供的引物进行PCR扩增。 PCR产物，然后与3个不同的限制性内切酶消化，安捷伦2100生物分析仪解决。在消化反应产生的片段长度可用于确定的DNA样本，准备从鱼的品种，使用RFLP图谱匹配软件包含实验衍生RFLP从相关的商业价值的鱼类物种模式的数据库。

Watch this Scientific Journal Video about DNA-based Fish Species Identification Protocol at JoVE.com

DNA-based Fish Species Identification Protocol

本刊物介绍了如何使用安捷伦鱼类识别系统，以确定提取DNA进行PCR和RFLP分析，鱼的种类。

以DNA为基础的鱼类物种鉴定协议

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

dna-based-fish-species-identification-protocol

Research

JoVE Journal

Biology

29.3K 次观看.  Agilent Technologies. 本刊物介绍了如何使用安捷伦鱼类识别系统，以确定提取DNA进行PCR和RFLP分析，鱼的种类。

视频: DNA-based Fish Species Identification Protocol

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

嗜中性粒细胞的分离协议

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

科研

生物学

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

非放射性原位挪威云杉杂交议定书适用的植物种类范围

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

荧光原位杂交（FISH）的议定书“在人类精子

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

侵入细胞DISC1蛋白种类的产生，纯化及表征

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

焦磷酸测序的微生物鉴定和表征

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

评估具体物种的贡献颅面开发利用鹌鹑，鸭嵌合体

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

使用软件和冷却装置进行鱼类精子评估

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...