Name: a fast silver staining protocol enabling simple and efficient detection of ssr markers using a non-denaturing polyacrylamide gel
Uploaded: 2018-04-20T13:30:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel at JoVE.com

这项工作由中国广东自然科学基金 (2015A030313500)、省级重点国际合作研究平台和广东省高等教育重点科研项目 (2015KGJHZ015)、科学与广东省烟草专卖管理技术方案 (201403、201705)、广东省科技计划 (2016B020201001)、全国大学生创新培训项目 (201711078001)。在本出版物中提及商号或商业产品, 纯粹是为了提供具体信息, 并不意味着美国农业部的建议或认可。美国农业部是一个平等机会提供者和雇主。

1. SSR 标记 PCR 产物的制备
<ol><li>准备所有的化学试剂和 PCR 反应, 包括模板 DNA (30 ng/µL), 2× pcr 大师组合 (包含 2× pcr 缓冲器, 每 dNTP 的0.4 毫米, 3 毫米氯化镁2, 0.1 U/µL Taq 脱氧核糖核酸聚合酶和染料), 10 µM 每向前和反向引物、蒸馏或去离子水 (dH2O)。 注: 本研究使用的 SSR 标记为 PT51333 (正向底漆序列: 5 '-GCACCTTTGGTTATCCGACA-3 ' 和反向引物序列: 5 '-TGCTTTAAGTCATGTACCAAATTGA-3 ') 和 PT50903 (正向底漆序列: 5 '-AAATTTCTTTCGGTGTGATAACTG-3 ' 和反向底漆序列: 5 '-AGAGACTGCCGTTTGATTTGA-3 ') 为烟草和 CX-43 (向前底漆序列: 5 '-TGGGGATGTGAGCTTCTTCT-3 ' 和反向引物 sequence:5 '-AGGGTTCCTTTGGGGTGATA-3 ') 和 CX-157 (向前底漆序列: 5 '-TCGACGCTGACTTCACTGAC-3 "和反向引物序列: 5 '-GGACAGCTTCACACATTTGC-3 ') 为开花大白菜。</li>
	<li>通过添加2µL 模板 DNA、5µL 2× pcr 总混合、每向前底漆和反向底漆0.2 µL 和 dH2.6O 的2µL, 制备10µL 的 pcr 组合物进行扩增。</li>
	<li>执行 PCR 放大反应在一个 thermocycler 的初始步骤94°c 为5分钟, 跟随38周期四十五年代在94°c 为变性, 四十五年代在55°c 为底漆退火并且1分钟在72°c 为引伸, 并且最后的延长步骤 10 min 在72°C;然后存储 PCR 产品在4°c, 供以后使用。</li>
</ol>2. 聚丙烯酰胺凝胶铸件溶液的制备
<ol><li>在 dh2o 中溶解54克的三基和27.5 克硼酸, 加入20毫升 (pH 0.5), 用 dh8.0o 将溶液调整为1、000毫升的最终体积, 5×1升。 注: 缓冲器可在室温下储存数月。</li>
	<li>将200毫升的5×缓冲液添加到 dH2O 到最终容积2升, 并在室温下贮存, 0.5×2升的缓冲液。</li>
	<li>在0.5×缓冲液中溶解29克分子生物学级丙烯酰胺和1克分子生物学级 n、n '-methylenebisacrylamide, 制备6% 的非变性聚丙烯酰胺凝胶溶液, 最终体积为500毫升。用铝箔盖住溶液瓶, 贮存在4摄氏度, 供以后使用。 注意: 丙烯酰胺被认为是一种强有力的神经毒素, 应该小心处理。在称量粉末和处理包含它的解决方案时, 总是戴上手套。</li>
	<li>通过将2克 ap 与 dH2O 溶为10毫升的最终体积, 准备20% 过硫酸铵 (aps) 溶液。它可以储存在-20 °c 几个月。 注: 为方便起见, 10 毫升 20% APS 溶液可 aliquoted 为1.5 毫升或2毫升的离心管储存在-20 摄氏度。在使用前要解冻并混合好。</li>
	<li>将3µL 的束缚硅烷溶于0.997 毫升的95% 乙醇中, 制备稀释的束缚硅烷溶液, 其中含有0.5% 的乙酸。它可以储存在4摄氏度的几个星期。 注意: 粘合硅烷是有毒的, 在处理溶液时戴上手套, 保持房间通风。</li>
</ol>3. 电泳用聚丙烯酰胺凝胶的制备
<ol><li>在自来水中用洗涤剂清洗一组玻璃板和垫片, 然后用 dH2进行彻底冲洗, 然后将这些盘子放在板材烘干机架上晾干。</li>
	<li>将1毫升的束缚硅烷溶液分散到矩形玻璃板的内表面, 干燥5分钟。 注: 如果粘合-硅烷溶液不均匀地分布在板材表面, 凝胶在染色过程中可能被分离, 导致染色效果不理想。</li>
	<li>用拭子将1毫升的排斥硅烷分散到缺口玻璃板的内表面, 用纸巾擦拭多余的排斥硅烷, 空气干燥5分钟。 注: 如果排斥硅烷不均匀地涂层玻璃板材的表面, 它可能通过部分剥离损坏胶凝体。</li>
	<li>在顶部装配有缺口板的玻璃板, 并用铸件夹具将装配的两侧夹紧。</li>
	<li>倒入适当数量 (40 毫升) 6% 非变性聚丙烯酰胺凝胶溶液的烧杯为33厘米宽 x 10 厘米高度和间隔厚度1.5 毫米, 添加20µL 的 tetramethylethylenediamine (TEMED) 和200µLfresh 20% APS 和混合轻轻。 注: 凝胶溶液的适量 (40 毫升) 是从两个板的内部容积 (30 厘米 x 8.5 厘米 x 0.15 厘米) 计算出来的。对于凝胶聚合的 TEMED 和 aps 的用量, 根据参考17, 有一个标准的凝胶溶液与 TEMED 和 aps 的比例。上面描述的凝胶溶液储存在4°c。如果凝胶溶液贮存在室温下, 应使用20µL 的 TEMED 和100µL 20% APS。用玻璃棒轻轻搅动凝胶溶液的混合物, 以避免溶液中气泡。</li>
	<li>立即将溶液倒入切口板边缘的组装玻璃板上, 将空间几乎填满, 然后插入梳子。在梳子上加入一小块凝胶溶液。 注: 保持玻璃板水平, 防止凝胶泄漏。如有必要, 用气泡钩去除气泡。</li>
	<li>允许凝胶在室温下设置为30分钟。 注: 聚合时间可能随着凝胶溶液和环境温度的变化而改变。</li>
	<li>在凝胶完全聚合后, 取下铸件夹具, 将所设的板套放在电泳槽单元中, 并将缺口板朝向上缓冲储层, 用大钳将板材固定在油箱单元上。</li>
	<li>将 0.5×1升的缓冲液倒入上、下腔。用吸管或注射器取出梳子, 用缓冲器彻底冲洗所有水井。 注: 确保在盘子底部的凝胶三明治完全浸泡在没有气泡的缓冲液中。</li>
</ol>4. 运行凝胶
<ol><li>将1µL 的 PCR 产物载入聚丙烯酰胺凝胶的各个井中。将 DNA 大小的梯子和 PCR 产品的样品一起载入凝胶的两侧。将安全盖固定在上部缓冲室。</li>
	<li>将引线连接到电源, 将颜色编码的红色与红色和黑色相匹配。在110伏的恒定电压下运行凝胶, 直到染料达到定义的位置, 通常为 ~ 70 分钟。 注: 电压和运行时间可根据 PCR 产品的大小进行调整。</li>
</ol>5. 用于检测非聚丙烯酰胺凝胶中 SSR 标记的银染色法
<ol><li>电泳后, 将缓冲液从上部腔体排出到大烧杯中通过阀门。分离侧夹, 从设备上取下盘子, 小心地将缺口玻璃板沿一侧与刮刀分开, 并确保该凝胶仍然附着在涂有束缚硅烷的其它玻璃板上。</li>
	<li>1 l 的新鲜浸渍溶液溶解1.5 克 AgNO3在 dh 的 1 L 中2o. 通过溶出毫升的氢氧化钠, 在 dh 1 O, 添加 10 ml 900 毫升甲醛的情况下, 制备2升的新开发溶液., 并调整为使用 dH2O 的最终卷1升。 注: 佩戴手套并小心处理上述化学品及解决方案。没有必要在黑暗中保留解决方案和相应的步骤。</li>
	<li>用足够的 dH2O 仔细冲洗凝胶和玻璃板, 去除电泳缓冲液, 然后将该板与凝胶置于塑料托盘上, 将凝胶浸入浸渍液的1升中。把托盘放在振动筛上。</li>
	<li>轻轻摇动托盘在 60 r/分钟3-4 分钟。 注: 在浸渍溶液中, 可根据硝酸银的凝胶厚度和浓度来调节震动时间。</li>
	<li>把盘子移出浸渍液, 放到另一个托盘上。用足够的 dH2O 两次, 分别从板表面和凝胶中冲洗残余浸渍液, 每双3-5 秒。</li>
	<li>将印版与胶面置于另一托盘上, 将板材浸入1升的开发溶液中, 然后轻轻地摇动托盘 50 r/分钟3分钟. 监测 dna 片段的出现和停止发育当 dna 片段信号的最高比率 对背景噪声被观察 (这步采取 ~ 3 分钟)。</li>
	<li>用足够的 dH2O 两次冲洗盘子和凝胶, 用纸巾将凝胶板烘干。</li>
	<li>使用适当的扫描仪扫描凝胶。300 dpi 的分辨率给出了 DNA 片段的清晰可视化。这种凝胶也可以用照相机拍照。</li>
	<li>SSR 标记的带状模式可以根据图像中的 DNA 片段进行评分。</li>
</ol>

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作者声明他们没有竞争的财政利益。

浸渍后的凝胶洗涤是一个关键步骤。洗涤时间和水量不足, 可能导致板材和凝胶表面的浸渍液不完全去除, 造成暗背景。适当的开发时间是另一个关键步骤, 过度开发可能导致黑褐色背景与低对比度图像的 DNA 片段。此外, 浸渍步骤显著影响 DNA 片段的染色效率。虽然在浸渍溶液中延长浸渍时间超过5分钟或增加 AgNO3量, 但不会显著改善染色质量, 将浸渍时间减少到少于3分钟或减少 AgNO3...

PCR 标记技术的发展使植物遗传学和育种学的研究发生了革命性的改变1。简单序列重复 (SSR) 标记是最常用和最多才多艺的 DNA 标记之一。其广泛的基因组覆盖率, 丰度, 基因组特异性和重复性是 SSR 标记的优点, 除了它们的呈共显性遗传检测异型基因型2。一些研究已经使用 SSR 标记来调查遗传多样性, 跟踪祖先, 构建基因连锁图, 并映射经济重要性状的基因3,4。
SSR 标记的 PCR 产物通常用琼脂糖或聚丙烯酰胺凝胶电泳进行分离, 然后用银染色或紫外光溴溴化物染色。聚丙烯酰胺凝胶中 DNA 片段的银染色比其他染色方法更敏感5,6 , 并已广泛用于检测 dna 片段, 如 SSR 标记7。
与许多生物实验室技术一样, 聚丙烯酰胺凝胶的银染色已稳步改善, 因为它第一次被报告为片段可视化技术在 1979年8。该技术最初被修改为检测 DNA 片段的巴萨姆et 等。6在 1991年, 然后由桑吉内蒂和同事在1994年改进9 。在最近几十年中, 该方法已进一步优化6、7、9、10、11、12、13、14,15. 然而, 大多数这些更新版本的协议仍然有一些缺点, 如高技术需求和长时间的固定和安装6, 这限制了这些协议的应用7, 11。为了在生物研究中常规应用银染色, 迫切需要采用低成本、高效的 DNA 片段检测相结合的最优协议。
此外, 聚丙烯酰胺凝胶可分为变性和非变性聚丙烯酰胺凝胶, 并可用于检测 SSR 标记使用银染色法。其效果和分辨率并无显著差异, 但非变性聚丙烯酰胺凝胶更易于处理, 且耗时较短16。
在以前的研究15的基础上, 本研究的目的是详细描述一个优化的银染色协议, 以便在非变性聚丙烯酰胺凝胶中快速、简便、低成本地检测 SSR 标记。

<table><tbody><tr><td>PCR master mix (Green Taq Mix)</td><td>Vazyme Biotech Co. Ltd, China</td><td>#P131-03</td><td></td></tr><tr><td>50-2000 bp DNA Ladder</td><td>Bio-Rad, USA</td><td>#170-8200</td><td></td></tr><tr><td>DL500 DNA marker</td><td>Takara Bio Inc., Japan</td><td>#3590A</td><td></td></tr><tr><td>Tris base</td><td>Sangon Biotech Shanghai, China</td><td>#77-86-1</td><td></td></tr><tr><td>Boric acid</td><td>Sangon Biotech Shanghai, China</td><td>#10043-35-3</td><td></td></tr><tr><td>EDTA-Na&lt;sub&gt;2&lt;/sub&gt;</td><td>Guangzhou Chemical Reagent Factory, China</td><td>#6381-92-6</td><td></td></tr><tr><td>Acrylamide</td><td>Sangon Biotech Shanghai, China</td><td>#79-06-1</td><td></td></tr><tr><td>N,N&#039;-methylene-bis-acrylamide</td><td>Sangon Biotech Shanghai, China</td><td>#110-26-9</td><td></td></tr><tr><td>N,N,N&#039;,N&#039;-Tetramethylethylenediamine</td><td>Sangon Biotech Shanghai, China</td><td>#110-18-9</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Guangzhou Chemical Reagent Factory, China</td><td>#7727-54-0</td><td></td></tr><tr><td>Bind-silane</td><td>Solarbio Beijing, China</td><td>#B8150</td><td></td></tr><tr><td>AgNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Beijing Co.,Ltd, China</td><td>#7761-88-8</td><td></td></tr><tr><td>Formaldehyde</td><td>Tianjin DaMao Chemical Reagent Factory, China</td><td>#50-00-0</td><td></td></tr><tr><td>NaOH</td><td>Guangzhou Chemical Reagent Factory, China</td><td>#1310-73-2</td><td></td></tr><tr><td>Acetic acid</td><td>Guangzhou Chemical Reagent Factory, China</td><td>#64-19-7</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>Tianjin DaMao Chemical Reagent Factory, China</td><td>#497-19-8</td><td></td></tr><tr><td>Ethanol</td><td>Guangzhou Chemical Reagent Factory, China</td><td>#64-17-5</td><td></td></tr><tr><td>HNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Guangzhou Chemical Reagent Factory, China</td><td>#7697-37-2</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;.5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm Chemical Reagent Beijing Co.,Ltd, China</td><td>#10102-17-7</td><td></td></tr><tr><td>Eriochrome black T(EBT)</td><td>Tianjin DaMao Chemical Reagent Factory, China</td><td>#1787-61-7</td><td></td></tr><tr><td>Plastic tray</td><td>Shanghai Yi Chen Plastic Co., Ltd, China</td><td>-</td><td></td></tr><tr><td>TS-1 Shaker</td><td>Qilinbeter JiangSu, China</td><td>-</td><td></td></tr><tr><td>BenQ M800 Scanner</td><td>BenQ, China</td><td>-</td><td></td></tr><tr><td>DYY-6C Power supply</td><td>Beijing Liuyi Instrument Factory, China</td><td>-</td><td></td></tr><tr><td>High throughout vertical gel systems, JY-SCZF</td><td>Beijing Tunyi Electrophoresis Co., Ltd, China</td><td>-</td><td></td></tr></tbody></table>

a fast silver staining protocol enabling simple and efficient detection of ssr markers using a non-denaturing polyacrylamide gel

简单序列重复 (SSR) 是植物和动物遗传研究和分子育种项目中最有效的标记之一。银染色是一种广泛应用于聚丙烯酰胺凝胶中 SSR 标记检测的方法。然而, 传统的银染色协议在技术上要求和耗时。与许多其他生物实验室技术一样, 银染色协议已稳步优化, 以提高检测效率。在这里, 我们报告一个简化的银染色方法, 大大降低试剂成本, 提高检测分辨率和图像清晰度。新的方法需要两个主要步骤 (浸渍和开发) 和三试剂 (硝酸银, 氢氧化钠和甲醛), 和只有7分钟的加工为非变性聚丙烯酰胺凝胶。与以前报告的协议相比, 这种新方法更容易、更快, 并且使用较少的化学试剂进行 SSR 检测。因此, 这种简单、低成本、有效的银染色协议将通过快速生成 SSR 标记数据, 有利于遗传图谱和标记辅助育种。

采用相应的 SSR 引物对 amplicons 甘蓝和烟草进行了 PCR 制备。电泳后, 采用上述银染色协议对聚丙烯酰胺凝胶进行染色, 明确检测到 SSR 标记的带状模式 (图 1)。
为了比较不同银染色协议的检测效率, 采用聚丙烯酰胺凝胶电泳法分离了烟草和开花白菜中 SSR 标记的 PCR 产物, 并用五的银染色方法进行了可视化。?...

Watch this Scientific Journal Video about A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel at JoVE.com

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

在这里, 我们报告一个简单和低成本的银染色协议, 只需三试剂和7分钟的处理, 并适合快速生成高质量的 SSR 数据的遗传分析。

一种快速的银染色协议, 能够使用非变性聚丙烯酰胺凝胶对 SSR 标记进行简单而有效的检测

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

a-fast-silver-staining-protocol-enabling-simple-efficient-detection

Research

JoVE Journal

Genetics

10.7K 次观看.  Guangzhou University. 在这里, 我们报告一个简单和低成本的银染色协议, 只需三试剂和7分钟的处理, 并适合快速生成高质量的 SSR 数据的遗传分析。

视频: A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

科研

生物化学

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the formation of a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in a solution of 1x (equivalent to 2.0 &#181;M) SYBR Green I (SG I) and 0.20 mM nitro blue tetrazolium that produces a purple precipitate of formazan when exposed to sunlight or specifically blue light. Also, DNA recovery tests were performed using an ampicillin resistant plasmid in an agarose gel stained with our method. A larger number of colonies was obtained with our method than with traditional staining using SG I with ultraviolet illumination. The described method is fast, specific, and non-toxic for DNA detection, allowing visualization of biomolecules to the "naked eye" without a transilluminator, and is inexpensive and appropriate for field use. For these reasons, our new DNA staining method has potential benefits to both research and industry.

This method allows selective staining and quantification of DNA in gels by soaking the gel in a SYBR Green I/Nitro Blue Tetrazolium solution and then exposing it to sunlight or a blue light source. This produces a visible precipitate and requires almost no equipment, making it ideal for field use.

基于蓝光照射诱发甲沉淀的 DNA 染色方法

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

在凝胶染色蛋白质

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

生物学

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

免疫与感染

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

聚丙烯酰胺凝胶中蛋白质的荧光银染色

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

通过后染色方法在聚丙烯酰胺凝胶中对芒果标记RNA的荧光可视化

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

聚丙烯酰胺凝胶电泳和银染色检测甘油糖

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Protein Analysis: A Technique to Separate and Visualize Proteins Based on Molecular Weight

Source: Sultana, S. et al. <a href="https://www.jove.com/v/62628">Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor</a>. J. Vis. Exp. (2021).
This video demonstrates a denaturing polyacrylamide-based separation technique for proteins. This technique helps in the preliminary identification of proteins based on their molecular weights.

用于蛋白质分析的十二烷基硫酸钠聚丙烯酰胺凝胶电泳:一种基于分子量分离和可视化蛋白质的技术

Source: Sultana, S. et al. Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor. J. Vis. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Fluorescent Silver Staining: A Technique to Visualize Total Protein in Polyacrylamide Gels

After electrophoresis, submerge the gel in a 100-milliliter solution containing ethanol and acetic acid on an orbital shaker, and incubate twice for 30 minutes each, at room temperature while shaking at 50 RPM. Next, wash the gel three times with ultrapure water in a clean container, with each wash lasting 10 minutes.
First, dissolve 0.01 grams of silver nitrate in 10 milliliters of ultrapure water to prepare a stock solution with a concentration of 0.1%. Add 100 microliters of this stock solution into 100 milliliters of ultrapure water to make the silver nitrate working solution. In a fume hood, submerge the gel in 100 milliliters of the silver nitrate working solution in a sealed glass chamber.
Use aluminum foil to protect the gel from light during impregnation, and incubate for 1 hour while shaking at 50 RPM on an orbital shaker. After this, wash the gel twice with ultrapure water in a clean container, with each wash using approximately 100 milliliters of water and lasting 60 seconds.
It is important to use ultrapure water to clean the gel after the silver impregnation step to minimize background staining.
First, add 50 milliliters of ultrapure water to 3 milligrams of TPE-4TA dye. Sonicate the solution for 3 minutes, and add 5 microliters of 1 molar sodium hydroxide solution in between each sonication session, to help dissolve the dye, usually up to three times. Then, check the fluorescence of the solution under a 365-nanometer UV lamp to ensure that the dye is fully dissolved. Only weak or non-emissive solutions indicate full dissolution.
To prepare the fluorogenic developing solution, add 10 milliliters of the TPE-4TA stock solution to 90 milliliters of ultrapure water. Use a pH meter to check the pH of the solution. Tune the solution to a pH between 7 and 9, using diluted sodium hydroxide solution or acetic acid, if the pH is out of range.
Next, transfer the gel to a clean and sealable container with 100 milliliters of the fluorogenic developing solution, and ensure that the gel is completely immersed. Seal the container, and cover it to protect it from light. Shake the container overnight on an orbital shaker at 50 RPM and at room temperature.
Transfer the gel to a clean container, and de-stain it in 100 milliliters of 10% ethanol for 30 minutes. Then, rinse the gel in ultrapure water for 5 minutes. The gel can be visualized on a benchtop trans-illuminator, or imaged on a gel documentation machine at the 365-nanometer channel or the 302-nanometer channel.