Name: agarose gel electrophoresis for the separation of dna fragments
Uploaded: 2012-04-20T12:00:00.000+00:00
Duration: 7 min 10 s
Description: Watch this Scientific Journal Video about Agarose Gel Electrophoresis for the Separation of DNA Fragments at JoVE.com

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>

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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

763.0K 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

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

通过智能手机实时跟踪DNA片段分离

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

科研

生物化学

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

生物学

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

用噻唑橙代替溴代或替代染料进行 DNA 电泳

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

DNA Gel Electrophoresis

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of agarose, and within the gel, charge particles will migrate and separate based on size. The negatively charged phosphates of the DNA backbone cause DNA fragments to move toward the anode - a positively charged electrode.
The video explains the mechanism by which DNA fragments are resolved on an agarose gel, and it provides a step-by-step generalized procedure for how to prepare agarose gels, load DNA samples, run a DNA gel, visualize DNA fragments, and properly dispose of the gel and running buffer after the experiment is concluded.

DNA Gel Electrophoresis: Concept, Procedure, and Applications

DNA凝胶电泳

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of ...

教育

JoVE Science Education

基础生物学

细胞分子生物学中的基本方法

Separating Protein with SDS-PAGE

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their size and nothing else. SDS, an anionic detergent, is used to produce an even charge across the length of proteins that have been linearized. By first loading them into a gel made of polyacrylamide and then applying an electric field to the gel, SDS-coated proteins are then separated. The electric field acts as the driving force, drawing the SDS coated proteins towards the anode with larger proteins moving more slowly than small proteins. In order to identify proteins by size, protein standards of a known size are loaded along with samples and run under the same conditions.
This video presents an introduction to SDS-PAGE by first explaining the theory behind it and later demonstrating its step-by-step procedure. Various experimental parameters, such as the polyacrylamide concentration and voltage applied to the gel are discussed. Downstream staining methods like Coomassie and silver stains are introduced, and variations of the method, like 2D gel electrophoresis are presented.

Separating Protein with SDS-PAGE: Procedure and Applications

用SDS-PAGE技术分离蛋白

Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis, or SDS-PAGE, is a widely-used technique for separating mixtures of proteins based on their ...

Gel Purification

Gel purification is used to recover DNA fragments after electrophoretic separation. DNA recovery from an agarose gel includes three basic steps: binding, washing and eluting from a silica column. DNA is believed to bind to silica in the presence of high salt via a salt bridge. Following binding, DNA is washed of impurities and eluted under low salt conditions disrupting this interaction.
This video goes through a step-by-step, generalized procedure for cutting out a band from the gel, gel solubilization, purification through binding to a silica column, and elution of purified DNA. In addition, the presentation discusses several tips for ensuring successful gel purification, including the importance of running an agarose gel with a marker or ladder that has DNA of known sizes.

Gel Purification: Recovery of DNA Fragments after Electrophoretic Separation

凝胶纯化

Gel purification is used to recover DNA fragments after electrophoretic separation. DNA recovery from an agarose gel includes three basic steps: binding, ...

DNA Agarose Gel Electrophoresis

Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify DNA fragments using a gel extraction protocol.
Gel extraction follows five major steps: running gel electrophoresis to separate fragments, isolating the individual bands, extracting DNA from those bands, and removing the dye and salts from the extracted mixture to obtain pure DNA.
In cloning experiments, both the insert and vector DNA fragments are obtained after digestion with restriction endonucleases. These DNA fragments of varying sizes are mixed with contaminants, such as enzymes, salts, etc., that can inhibit the ligation reaction that follows. Gel extraction is therefore used to obtain pure DNA fragments before ligation.
To set up a gel for extraction, a lower percent (0.7-0.8%) agarose solution is used to ensure efficient migration of the DNA bands. In addition, a wide-combed gel cast is used to obtain thick DNA bands that are easy to isolate. Then, gel electrophoresis is performed at a lower voltage to prevent heating of the gel and damage to the DNA.
Once gel electrophoresis is complete, the ethidium bromide-stained DNA is identified by exposing the gel to long-wavelength UV for a short time. Short exposure to UV radiation prevents damage to DNA. This is followed by cutting out the desired band using a clean razor blade.
The isolated gel slice containing the DNA fragment of interest is then processed through one of the commercially available gel extraction kits. All of these kits follow the same basic principle. First, the agarose gel is dissolved by a buffer solution containing salts, such as sodium iodide. Next, DNA is bound to a column containing an anionic resin and washed a few times with a dilute alcoholic solution to remove impurities. The column is then dried by spinning in a centrifuge. The pure DNA can now be eluted with buffer or deionized water.

Agarose Gel Electrophoresis for Separation of DNA Fragments

DNA 琼脂糖凝胶电泳

Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify ...

JoVE Core

分子生物学

Unit 2

Studying DNA and RNA

关键术语与概念

Two-Dimensional Semi-Denaturing Agarose Gel Electrophoresis: A Technique to Separate Polymorphic Amyloids Fibers Based on Size Heterogeneity

To prepare the gel, add 2 grams of agarose powder to 200 milliliters of TAE buffer in a glass beaker. Then, heat the beaker in a microwave to melt the agarose.
Next, add 1 milliliter of 20% SDS to a final concentration of 0.1%. Gently swirl the beaker.
Next, pour the liquid agarose on a 15 cm x 14 cm gel slab. To remove any air bubbles, use a 1-milliliter pipette. Then, place a 20-well comb on top of the gel.
Next, add approximately 60 micrograms of whole-cell lysate in the far-right lane of the gel. Run the gel at 60 volts for about four hours, using the TAE buffer containing 0.1% SDS, as the running buffer.
To run the gel in the second dimension, carefully rotate the gel by 90 degrees counter-clockwise. Then, run the jail at 60 volts for about four hours.

二维半变性琼脂糖凝胶电泳:一种基于尺寸异质性分离多态性淀粉样蛋白纤维的技术

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

EoE Sub Articles

Agarose Gel Electrophoresis of DNA Amplicons Post PCR: A Method to Analyze Products of Multiplex PCR and Evaluate PCR Reaction Success

According to the manufacturer&#39;s recommendations, add agarose gel powder in 1x TBE buffer to reach 1.5 (weight/volume) percent, and heat to dissolve.
Prior to casting, tilt the dissolved gel solution briefly under running water. Add 5 microliters of fluorescent nucleic acid dye per 50 microliters of gel solution, and mix.
Assemble and level the casting tray. Add combs as appropriate for the number of samples. Pour the gel solution into the cast, and wait for it to solidify.
After that, submerge the cast containing the gel in 1x TBE buffer in a gel electrophoresis system. Mix 5 microliters of the PCR products together with 2 microliters of loading dye in new PCR strips. Transfer 5 microliters of the mixtures to the wells in the gel. Save at least one well empty. Add 5 microliters of DNA ladder in the empty well for reference.
Plug in the electrodes and run the gel at 110 volts per 15 centimeters for approximately 1 hour. Use a UV-based gel imaging system to verify the presence of multiple bands representing PCR amplicons.

PCR 后 DNA 扩增子的琼脂糖凝胶电泳:一种分析多重 PCR 产物并评估 PCR 反应成功的方法

Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight

To begin this procedure, add 250 microliters of a previously prepared protein sample solution to each slot of a 13-centimeter IPG strip holder. Place an 18-centimeter IPG strip, gel-side down, onto each protein sample solution, taking care to avoid bubbles. Then, add 3 to 4 milliliters of mineral oil to cover each IPG strip.
Next, assemble each IPG strip holder into an isoelectric focusing system, with the pointed end on the back plate, and the square end on the front plate. After rehydrating the IPG strips for 18 hours at room temperature, set the IEF parameters on the isoelectric focusing system with a total runtime of 7.5 hours and 36,875 volt hours.
After IEF, take out each IPG strip from the isoelectric focusing system, and remove the excess mineral oil with an insoluble paper towel. Then, wrap each strip in a sheet of plastic wrap, and store at -80 degrees Celsius.
To cast a 12% PAGE gel, add double-distilled water, 1.5 molar Tris-hydrochloric buffer, 40% acrylamide/bisacrylamide stock solution, 10% ammonium persulfate, and TEMED to a beaker. Mix the gel solution gently, taking care to avoid bubbles.
Gently pour the gel solution into a multi-casting chamber, up to the expected gel height of 19 centimeters. Immediately, add about 3 milliliters of double-distilled water to the multi-casting chamber to cover each resolving gel, and allow each gel to polymerize for about one hour.
This step is very important to prepare a high-quality SDS-PAGE gel. An equal volume water must be immediately added on the top of each gel.
Prepare two liters of electrophoresis buffer, then, add the buffer to an electrophoresis separation unit buffer tank.
After removing the focused IPG strips from the freezer, place them in a plastic vessel containing 1 milliliter of reducing equilibrium buffer and a trace of bromophenol blue, and equilibrate them for 10 minutes with gentle rocking. After removing the reducing equilibrium buffer, add 4 milliliters of alkylation equilibrium buffer, and a trace of bromophenol blue to the IPG strips, and equilibrate them for 10 minutes with gentle rocking.
During gel equilibrium, disassemble the multi-casting chamber, and remove the prepared resolving gel cassette. Rinse the gel cassette three times with double-distilled water, then, remove excess water from the gel cassette with an insoluble paper towel, and place it in a gel stander.
Following this, rinse the equilibrated IPG strips with electrophoresis buffer, and remove excess liquid on each IPG strip surface with an insoluble paper towel. Place an IPG strip onto the resolving SDS-PAGE gel, allowing the IPG strip&#39;s plastic side to contact the longer glass plate, with the pointed end to the left.
Now, quickly add 3 to 4 milliliters of hot 1% agarose and SDS electrophoresis buffer, to seal the IPG strip on the top of each SDS-PAGE gel, and place the top side of the IPG strip down on the top of the shorter glass plate, taking care to avoid bubbles.
These steps are very important to seal the IPG strip to an SDS-PAGE gel. The hot agarose solutions must be immediately added, and the IPG strip must be immediately placed into the top of the SDS gel.
After polymerization, insert the assembled gel cassette vertically between the plastic gaskets and a vertical electrophoresis system. Place the top of the gel with the IPG strip next to the cathode. Next, adjust the level of electrophoresis buffer to immerse the gel cassette by adding the appropriate volume of buffer to the electrophoresis system.
Connect and set the power supply in constant voltage mode, and run at 200 volts for about 370 minutes while monitoring the dye. After the run is complete, take the gel cassette out of the electrophoresis system and gently remove the gel from the gel cassette, taking care to avoid tearing the gel.