1。凝胶的制备<ol><li>称量烧瓶的琼脂糖适当质量。琼脂糖凝胶电泳准备使用AW / V比例解决方案。琼脂糖凝胶浓度的DNA片段的大小将取决于与大多数凝胶0.5％-2％之间不等，要分开。缓冲区的数量不应该超过1/3瓶的容量更大。 </li><li>添加运行缓冲液的琼脂糖含瓶。涡流混合。运行缓冲区的最常见的凝胶栓塞（三醋酸40毫米，1毫米EDTA）和TBE（45毫米的Tris-硼酸，1毫米EDTA）。 </li><li>融化的琼脂糖/缓冲液。这是最常见的微波炉加热，但也可以通过本生灯火焰。在间隔30秒，取出拌匀瓶和漩涡的内容。重复，直到琼脂糖完全溶解。 </li><li>溴化乙锭（EtBr）可添加浓度为0.5微克/毫升。另外，也可能被凝胶染色后电电泳在运行缓冲液含0.5微克/毫升乙锭为15-30分钟，其次是在运行的时间长度相等的缓冲区脱色。 </li></ol>注：乙锭是一种可疑的致癌物，必须妥善处置每机构管理条例。处理凝胶含有ETBR时，应始终戴手套。可替代染料染色的DNA ETBR但仍然是最流行的一种，由于其敏感性和成本。 <ol start="5"><li>允许琼脂糖在65℃水浴中冷却，无论是在台式或由孵化。如果不这样做，将凝胶托盘变形。 </li><li>凝胶托盘放到铸造设备。另外，也可能磁带凝胶的托盘，以创建一个模具的开放边。凝胶模具放到一个适当的梳子，以创建井。 </li><li>倒入熔化的琼脂糖凝胶模具。让琼脂糖设置在室温。取出梳子和凝胶放置在凝胶中。另外，在GEL也可以被包裹在保鲜膜，并保存于4°C，直到使用（ 图1）。 </li></ol> 2。设立仪器凝胶DNA片段分离<ol><li>添加载入染料要分开的DNA样本（ 图2）。凝胶上样染料通常由的6X浓度（0.25％，0.25％溴酚蓝二甲苯青，30％甘油）。装载染料有助于跟踪已走过多远，您的DNA样本，还允许陷入凝胶样品。 </li><li>方案的电源供应器所需的电压（电极之间1-5V/cm）。 </li><li>加入足够的运行缓冲液，凝胶覆盖面。重要的是要使用相同的运行缓冲液作为一个用于制备凝胶。 </li><li>凝胶盒的导线连接电源。打开电源和验证，胶盒和电源工作。 </li><li>取下盖子。慢慢地而谨慎地装载到凝胶的DNA样本（S）（ 图3）。一个相应的DNA大小标记应始终被载入随着实验样本。 </li><li>更换凝胶盒的盖子。阴极（黑线）应该接近井比阳极（红色导线）。仔细检查，电极插入到正确的插槽电源。 </li><li>接通电源。运行凝胶，直到染料迁移到适当的距离。 </li></ol> 3。观察分离的DNA片段<ol><li>当电泳完成后，关闭电源，取出凝胶盒的盖子。 </li><li>从凝胶中取出凝胶。从凝胶表面，排出多余的缓冲区。将凝胶托盘上的纸巾吸收任何额外的运行缓冲。 </li><li>从凝胶托盘取出凝胶和暴露在紫外线照射下，凝胶。这是最常见的使用凝胶文件系统（ 图4）。 DNA条带应显示为橙色荧光带。采取的凝胶图片（ 图5）。 </li><li>妥善处置每机构管理条例凝胶和运行缓冲区。 </li></ol> 4。代表结果 图5表示一个典型的结果后，琼脂糖凝胶电泳PCR产物。分离后，所产生的DNA片段作为明确界定波段可见。 DNA标准或梯子应允许有用的样品带的大小确定，在一定程度上分离。在这个例子所示，765基点，880 bp和1022 bp的DNA片段的1.5％琼脂糖凝胶电泳分离，连同2日志的DNA梯状条带。 <img alt="图1。" src="/files/ftp_upload/3923/3923fig1.jpg" /> 图1。琼脂糖凝胶固化后去除梳子。 <img alt="图2。" src="/files/ftp_upload/3923/3923fig2.jpg" /> 图2。样染料学生加入到她的DNA样本。 <img alt="图3。" src="/files/ftp_upload/3923/3923fig3.jpg" /> 图3。学生装成凝胶以及DNA样本。 <img alt="图4。" src="/files/ftp_upload/3923/3923fig4.jpg" /> 图4。凝胶文件系统的一个例子。 <img alt="图5。" src="/files/ftp_upload/3923/3923fig5.jpg" /> 图5。凝胶后电泳图像。 ，ETBR被添加到凝胶电泳至0.5微克/毫升的终浓度为1小时，在100至五分离前。凝胶被暴露在紫外线和凝胶文件系统所采取的图片。

<ol>
	<li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. , (2001).</li><li>Kirkpatrick, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+agarose+gel+properties.">Overview of agarose gel properties.</a> Electrophoresis of large DNA molecules: theory and applications. , 9-22 (1991).</li><li>Helling, R. B., Goodman, H. M., Boyer, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+endonuclease+R&#8226;EcoRI+fragments+of+DNA+from+lambdoid+bacteriophages+and+other+viruses+by+agarose-gel+electrophoresis.">Analysis of endonuclease R&#8226;EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis.</a> J. Virol. 14, 1235-1244 (1974).</li><li>Smith, S. B., Aldridge, P. K., Callis, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+individual+DNA+molecules+undergoing+gel+electrophoresis.">Observation of individual DNA molecules undergoing gel electrophoresis.</a> Science. 243, 203-206 (1989).</li><li>Aaji, C., Borst, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gel+electrophoresis+of+DNA.">The gel electrophoresis of DNA.</a> Biochim. Biophys. Acta. 269, 192-200 (1972).</li><li>Lai, E., Birren, B. W., Clark, S. M., Simon, M. I., Hood, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+field+gel+electrophoresis.">Pulsed field gel electrophoresis.</a> Biotechniques. 7, 34-42 (1989).</li><li>Devor, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IDT tutorial: gel electrophoresis. , (2010).</li><li>Serwer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gels:+properties+and+use+for+electrophoresis.">Agarose gels: properties and use for electrophoresis.</a> Electrophoresis. 4, 375-382 (1983).</li><li>Dea, I. C. M., McKinnon, A. A., Rees, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tertiary+and+quaternary+structure+and+aqueous+polysaccharide+systems+which+model+cell+wall+adhesion:+reversible+changes+in+conformation+and+association+if+agarose,+carrageenan+and+galactomannans.">Tertiary and quaternary structure and aqueous polysaccharide systems which model cell wall adhesion: reversible changes in conformation and association if agarose, carrageenan and galactomannans.</a> J. Mol. Biol. 68, 153-172 (1972).</li><li>Sharp, P. A., Sugden, B., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+two+restriction+endonuclease+activities+in+H.+parainfluenzae+using+analytical+agarose-ethidium+bromide+electrophoresis.">Detection of two restriction endonuclease activities in H. parainfluenzae using analytical agarose-ethidium bromide electrophoresis.</a> Biochemistry. 12, 3055-3063 (1973).</li></ol>

我们什么都没有透露。

已被证明是琼脂糖凝胶电泳分离核酸的高效和有效的方式。琼脂糖凝胶强度高，允许大DNA片段的分离处理的比例很低凝胶。分子筛是由7琼脂糖凝胶基质束所产生的毛孔大小。在一般情况下，较高浓度的琼脂糖，孔径越小。传统的琼脂糖凝胶电泳在100 bp和25 kb的DNA片段之间的分离是最有效的。分开大于25 KB的DNA片段，将需要使用脉冲场凝胶电泳，其中包括目前从两个不同的方向交替应用。在这样的较大规模的DNA片段分离的速度，他们在重新调整自己与电流方向的变化。 DNA片段小于100个基点，更有效地使用聚丙烯酰胺凝胶电泳分离。不像琼脂糖凝胶电泳，聚丙烯酰胺凝胶基质是通过自由基驱动化学反应形成的。这些稀释剂的凝胶浓度较高，运行和垂直方向有更好的分辨率。在现代的DNA测序毛细管电泳，使毛细管用凝胶基质填充。毛细管允许使用高电压的应用，从而使分离的DNA片段（DNA序列的测定）迅速。 琼脂糖可以修改，创建羟乙基低熔点琼脂糖。低熔点琼脂糖一般是分离的DNA片段需要隔离时使用。羟乙基降低琼脂糖包的堆积密度，有效地降低其孔径8。这意味着一个同样大小的DNA片段，将需要更长的时间将通过低熔点琼脂糖，而不是一个标准的琼脂糖凝胶电泳凝胶。因为彼此关联的束通过非共价相互作用9，它有可能重新融化的琼脂糖凝胶电泳后，它已设置。 ETBR是最常见的试剂，用于染色的DNA琼脂糖凝胶10。当暴露在紫外线下，乙锭分子芳香环中的电子被激活，从而导致释放的能量（光）电子返回基态。 ETBR工程通过插层本身在DNA分子中的浓度依赖性。这允许根据其强度在任何特定的DNA带的DNA量的估计。由于其正电荷，ETBR使用减少15％的DNA迁移率。 ETBR是一个可疑的诱变剂和致癌物质，因此必须小心处理琼脂糖凝胶含有它时。 ETBR此外，被认为是危险废物，必须妥善处置。 DNA琼脂糖凝胶替代污渍的SYBR黄​​金的SYBR绿，结晶紫和甲基蓝。其中，甲基蓝和结晶紫不需要暴露的DNA条带的可视化的紫外光凝胶，如果需要恢复从凝胶中的DNA片段，从而减少了基因突变的概率。然而，他们的敏感性比ETBR较低。 SYBR黄​​金和SYBR GREEN是高度敏感，防紫外线依赖低于ETBR毒性染料，但它们是相当昂贵的。此外，所有的替代染料要么不能或不工作时，直接添加到凝胶，凝胶电泳后染色后。由于成本，易用性和敏感性，ETBR仍有许多研究者的首选染料。然而，在某些情况下，如危险废物处置时很难或青年学生时，正在执行一项实验，毒性较低的染料可能被优先考虑。 载入染料在凝胶电泳为三个主要目的。首先，他们增加密度的样品，允许它沉入凝胶。第二，染料提供颜色和简化加载过程。最后，按标准费率的染料移动通过凝胶，使DNA片段的迁移距离的估计。 分离DNA片段的确切大小，可确定策划针对每个波段行驶距离的DNA标准的不同频段的分子量日志。 DNA标准包含一个预先确定大小的DNA片段，可以对未知的DNA样本相比的混合物。重要的是要注意，不同形式的DNA以不同的速率通过凝胶移动。超螺旋质粒DNA，由于其结构紧凑，通过凝胶最快的移动，循环行驶最慢的开放形式，一个同样大小的线性DNA片段。 总之，通过在20世纪70年代以来琼脂糖凝胶DNA分离，它具有被证明是最有用和最通用的技术，在生物科学的研究之一。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>琼脂糖我</td><td> AMRESCO </td><td> 0710 </td><td></td></tr><tr><td>硼酸</td><td>西格玛</td><td> B7901 </td><td></td></tr><tr><td>溴酚蓝</td><td>西格玛</td><td> B8026 </td><td></td></tr><tr><td> EDTA的</td><td>西格玛</td><td> E9884 </td><td></td></tr><tr><td>溴化乙锭</td><td>西格玛</td><td> E7637 </td><td>致癌，必须作为危险废物处置</td></tr><tr><td>冰醋酸</td><td>费舍尔</td><td> BP2401-212 </td><td>腐蚀性的</td></tr><tr><td>甘油</td><td>费舍尔</td><td> G33-1 </td><td></td></tr><tr><td> xylenécyanol FF </td><td>西格玛</td><td> X4126 </td><td></td></tr><tr><td> Tris碱</td><td>西格玛</td><td> T1503 </td><td></td></tr><tr><td>调查/ FX凝胶文件系统</td><td> fotodyne </td><td></td><td></td></tr><tr><td>猫头鹰Easycast B1微型凝胶电泳系统</td><td> Thermo Scientific的</td><td> B1-的PTM </td><td></td></tr><tr><td>每股盈利301电源</td><td> GE医疗集团</td><td> 18-1130-01 </td><td></td></tr></tbody></table>

agarose gel electrophoresis for the separation of dna fragments

琼脂糖凝胶电泳分离DNA片段的不同大小不等，从100个基点，25 KB 1是最有效的方法。琼脂糖是从海藻属石花菜和江蓠 ，分离和重复agarobiose（L-和D-半乳糖）亚基2组成。凝胶过程中，关联的琼脂糖凝胶聚合物非共价和束的孔径确定凝胶的分子筛性能形成一个网络。使用琼脂糖凝胶电泳，彻底改变了DNA的分离。在此之前通过琼脂糖凝胶，DNA主要是分离采用蔗糖密度梯度离心，只提供了一个大小近似。使用琼脂糖凝胶电泳分离DNA，DNA被装入预制凝胶和当前应用井。磷酸骨架的DNA和RNA分子是带负电荷，因此放置在电场中时，DNA片段，将迁移到p阳极ositively收费。由于DNA有一个统一的质量/电荷比，DNA分子在琼脂糖凝胶电泳分离模式行驶距离成反比，其分子量3日志的大小。 DNA通过琼脂糖凝胶电泳运动的领导模式是“偏爬行”，即领先前进和拉其余的分子沿4。 DNA分子通过凝胶迁移率取决于以下几点：1）DNA分子的大小; 2）琼脂糖浓度; 3）DNA构象; 4）电压应用，5）存在的溴化乙锭，6）型琼脂糖和7）电泳缓冲液。分离后，DNA分子的紫外光照射下，可以可视化后，以适当的染料染色。按照此协议，学生应该能够： <ol><li>了解DNA片段在凝胶基质中分离机制</li><li>了解如何构DNA分子将决定其流动性，通过凝胶基质</li><li>他们的需要，确定适当浓度的琼脂糖溶液</li><li>琼脂糖凝胶电泳的DNA样本准备</li><li>成立凝胶电泳仪电源</li><li>选择适当的电压为分离的DNA片段</li><li>了解溴化乙锭的DNA条带的可视化的机制，允许</li><li>确定分离的DNA片段的大小</li></ol>

Watch this Scientific Journal Video about Agarose Gel Electrophoresis for the Separation of DNA Fragments at JoVE.com

Agarose Gel Electrophoresis for the Separation of DNA Fragments

描述一个基本协议的DNA片段，用琼脂糖凝胶电泳分离。

琼脂糖凝胶电泳分离DNA片段

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

agarose-gel-electrophoresis-for-the-separation-of-dna-fragments

Research

JoVE Journal

Biology

759.3K 次观看.  University of California Los Angeles. 描述一个基本协议的DNA片段，用琼脂糖凝胶电泳分离。

视频: Agarose Gel Electrophoresis for the Separation of DNA Fragments

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio film communities. So I am profiling the bacterial communities by looking at their 60 N sequence and how this sequence changes over time in space. And to do that, I'm using DDGE.This is not a normal acry of my well, it needs to be kept at 60 degrees when it's run and the buffer needs to be pumped continuously. So we're gonna warm up the buffer for about an hour until it's at 60 degrees, and then we can run the towel. Now I'm gonna wash this plate with dilute uc solution, peeling around for impurities, andone, light that up.Anything like that can cause regularities. It is important to wipe off all the water. So when I assemble the gel, I'll put that chip on the outside of the gel.So the gel will be in between these two pieces of glass and the chip will be on the outside and it won't affect the gel in between. So now I'm gonna assemble the gel in these clamps and then I squeeze them together and tighten by turning to the right. As with any gel, it's important to make sure that the bottom is flush.To prevent making, we now turn the gel around and put it in here to get sealed with the gasket. So it's important when you do this that the gel, that the plates are actually lined up with the gasket here. So these knobs on the side then go in and you press down to type the seal.So this is a 30 mil syringe. And this part and this part come with the bio red kit and it just on like this. And that's just to get rid of the excess water in the tubes.So put the tubing onto the gel to load the gel. First you put an adapter into the tubing and then a needle look with that small adapter and put it right in the center of the gel just so it's fairly below the bottom glass. Then this wire adapter goes into the long tubing and it's going to connect to equal syringes containing the high and the low density solution.So these are pre-prepared solutions. The blue one, I've added a dye here that just indicates that this is the most denaturing solution and this is the least fitting train solution, the low density one. And they have everything in them.And now I'm gonna to make, gonna be Actually I twice as much the week. And once you've got the tempt, you have about five to seven minutes to fill the gel before the colon rises. And now we fill the syringe juice with the solution.So I'm gonna fill it with about 15 the solution. And then to get rid of any air bubbles, I'll turn it upside down and squeeze gently until that top air bubble is gone. Now this little screw here is gonna go into this groove.This adapter is gonna line up with this clamp here, and it's gonna get C clamped in very tightly. And then this tubing is gonna attach to the syringe. And the same thing is done on the other side with the high density matrix where it's time to deliver.And so I'm gonna be turning this wheel in a steady clockwise motion. And first the blue solution is gonna go out first because that's the the most in nature, and it's gonna form the bottom part of the gel. And then after a little while, the clear solution will start filling in the gel and the Gradient will be formed.One moment is the gently on an angle to And as soon as you finish pouring the gel to wash out your tubes immediately because they will polymerize Going, It's the group there. And then press down. So this little, And on the other side, I'll do the same thing.Okay.Hi, my name is Celeste Peterson and I'm a fellow at Harvard University studying microbial bio communities. So I am profiling the bacterial communities by looking at their 60 F sequence and how the sequence changes over time in space. And to do that, I'm using DDGE.So DGE is Dena gel electrophoresis. And basically the idea is that there's a gradient and the DNA, as it runs down the gradient gets de natured. And so different 16 species will stop at different points along the gradient.And so it ends up being a fingerprint of the different sexiness species in the community. So the goal of my experiment is to see who is in the community and how that's changing over time and space. And I can run the sexiness DDD gel and then I can cut out the bands and identify the species that are represented in each community.So one of the biggest issues when, when doing A DDG gel is to figure out the gradient that's the best. And so that is just something that needs to be played around with and figuring out for a particular community which type of gradient. So the one I'm using today is 35 to 55%urea.One of the big problems that can happen with this technique is that smears come up in in the leans. And I found for that, for whatever reason, one of the best things to do is to run the gels for a really long time and then the smears disappear. Well, today I'm just using it for general bacterial communities, but this can also be used for a specific family of bacteria to see how those change over time, and those tend to be very clean gels.You can also look at mutations in single genes with DDGE.

变性梯度凝胶电泳（DGGE）

科研

生物学

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

优化的纤维蛋白凝胶珠检测血管生成的研究

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

使用琼脂糖凝胶为基础的微流体装置的评估神经干细胞的运动

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

半变性洗涤剂琼脂糖凝胶电泳筛选淀粉样蛋白聚合

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

变性尿素聚丙烯酰胺凝胶电泳（尿素页）

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Electroeluting DNA Fragments

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.

This procedure allows the purification of DNA fragments with high yield.

Electroeluting的DNA碎片

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-SEQ：蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...