琼脂糖凝胶电泳.ppt

2019/6/17,1,Agarose Gel Electrophoresis 琼脂糖凝胶电泳,2019/6/17,2,After the experiment is finished, students should know how to make an agarose gel.,掌握琼脂糖凝胶电泳的操作的方法。,一、实验目的 (Experimental Purpose),2019/6/17,3,Gel electrophoresis,2019/6/17,4,二、实验原理(Experimental Principle),Electrophoresis is a technique used to separate and sometimes purify proteins and nucleic acids, which differ in size, charge or conformation from each other. Electrophoresis has become the most widely used techniques in biochemistry and molecular biology.,电泳常用于分离和纯化那些分子大小、电荷性状或分子构象有所不同的生物大分子尤其是蛋白质和核酸。正因为如此，电泳已成为生物化学和分子生物学中应用最为广泛的技术之一。,2019/6/17,5,Agarose is a polysaccharide extracted from seaweed. Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques.,琼脂糖是一种海藻多糖，琼脂糖胶分离范围很大，但其分辨率却相对较低。通过改变琼脂糖凝胶的浓度，应用标准的电泳技术可以分离200到50,000 bp 大小的 DNA 片断。,琼脂糖凝胶浓度越大，凝胶就越硬。较高浓度的琼脂糖胶有利于较小的DNA片断分离，而较低浓度的琼脂糖胶则可以分离较大的DNA片断。,The higher the agarose concentration, the “stiffer“ the gel. Higher concentrations of agarose help in the separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.,2019/6/17,6,Agarose is typically used at concentrations of 0.5% to 2%.,The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes.,琼脂糖胶浓度一般在0.5到2之间。,通过观察示踪染料的迁移距离可以判断DNA的迁移距离。溴酚蓝染料在琼脂糖凝胶的迁移速率最大。,当迁移足够距离后，就可以通过Gelview染色来观察DNA片断。,When adequate migration has occured, DNA fragments can be visualized by staining with Gelview.,2019/6/17,7,2019/6/17,8,Gelview is a fluorescent dye. It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of Gelview. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator.,Gelview是一种荧光染料。它可以在做胶时混入其中在电泳时进行染色，也可以待电泳完成后将凝胶浸泡在稀释的Gelview 溶液中进行染色 。但必须将凝胶置于紫外透射仪中才可以对凝胶中的DNA或RNA进行观察。,2019/6/17,9,三、试剂与器材（Reagents and apparatus）,1. Agarose. 2. TBE 5 stock solution (1 liter): 54 g Tris base, 27.5 g boric acid, 20ml 0.5 M EDTA, pH 8.0. 3. 10 loading buffer: 0.25% bromophenol blue, 40% sucrose in water. 4. Equipment: beaker, graduated cylinder, stir bar, microwave, Pan balance, comb, electrophoresis tank, and Electro-phoresis System , Ultraviolet transilluminator. 5. Gelview,2019/6/17,10,. Preparation of the gel （凝胶的制备） 1.制备1琼脂糖凝胶。称取0.5g琼脂糖，放人锥形瓶中，加入50mL的 0.5TBE 缓冲液，放入微波炉加热至完全溶化，则为1琼脂糖凝胶液。（由于蒸发作用，溶解前在容量瓶上作一个记号，溶解后用三蒸水补足）,四、实验步骤(Experimental Procedures),2019/6/17,11,2.制胶器的安装 取多功能制胶器，洗净，晾干； 将多功能制胶器放置于一水平位置，选择126cm制胶架，然后选择1.5mm 18teeth的梳子（最大加样量25l）； 将所选择规格的梳子插入制胶架的定位槽中。,2019/6/17,12,3. 将熔化的琼脂糖凝胶液转入Gelview专用的三角瓶中，然后加入Gelview 5l。 4. 将冷到60左右的琼脂糖凝胶液，缓缓倒入所选择的制胶槽内，直至有机玻璃板上形成一层均匀的胶面(注意不要形成气泡)。 5. 待胶凝固后（30-60min），轻轻拔掉梳子，将凝胶盘从制胶槽中取出，放入电泳槽内。 6. 加入电泳缓冲液(0.5)至电泳槽中。,2019/6/17,13,. Loading DNA samples (加样),用移液枪缓慢将DNA样品垂直加入加样孔直至开口下方。 1.DNA samples ： 10l 2. Loading buffer (6X)：2l 3. DNA markers ：6l,Total 12l,2019/6/17,14,.Gel（电泳）,1. 接通电泳槽与电泳仪的电源(注意正负极，DNA片段从负极向正极移动)。保持电压 6080 V。 2. 当溴酚蓝染料移动到距凝胶前沿1-2cm处，停止电泳。,2019/6/17,15,. Gel Interpretation （凝胶图像解释）,The uncut DNA lane may have several bands in it. This occurs because the mobility of plasmid DNA in an agarose gel depends on its molecular conformation as well as its size in base pairs. Plasmid DNA can exist in any one of three major conformations:,未切割的质粒DNA在其泳道上也许会出现几个条带，之所以这样是由于质粒DNA在琼脂糖凝胶中的迁移距离是由其分子构象及其碱基对大小所决定的。质粒DNA以下列三种主要构象中的任何一种形式存在：,2019/6/17,16,Supercoiled - plasmid is usually seen as a supercoil in a bacterial cell. This form of the plasmid will move the fastest through the gel due to its compact structure.,超螺旋质粒在细菌细胞以超螺旋结构存在，由于其结构致密，它在凝胶中的泳动速度最快。,2019/6/17,17,Nicked-During plasmid DNA replication, topoisomer-ase I introduces a nick into one strand of the DNA helix and uncoils the plasmid. Physical shearing and enzymatic cleavage during plasmid isolation may also introduce nicks into the supercoiled plasmid to produce a relaxed open circular structure.,切口在质粒DNA复制过程中，拓扑异构酶I会在DNA双螺旋中的一条链中引入一个切口，解开质粒的超螺旋。在质粒分离过程中由于物理剪切和酶的切割作用同样也会在超螺旋质粒中引入切口从而产生松散的开环结构。这种形式的质粒迁移速率介于超螺旋和线性DNA之间。,2019/6/17,18,LinearLinear plasmid DNA occurs when damage results in strand nicks directly opposite each other on the DNA helix. This form is the slowest migrating form of plasmid. The presence of linear DNA in a plasmid preparation is a sign of either nuclease contamination or sloppy lab procedure.,线性当DNA损伤在DNA双链相对应的两条链上同时产生切口时，就会出现线性质粒DNA，这种DNA的泳动速率最慢，质粒制备过程个出现线性DNA说明存在核酸酶污染或实验操作有问题。,2019/6/17,19,五、实验结果 (experimental results),1、DNA相对分子质量标准物（maker） 2、pUC19质粒DNA经EcoRI完全酶解 3、 pUC19质粒DNA经EcoRI部分酶解 4、自提的pUC19质粒DNA（此结果提得较好，为1条带，以超螺旋为主。）,2019/6/17,20,六、注意事项(Notes),The mobility of the DNA depends on the following factors： Molecular size of DNA DNA的分子大小分子量越小，迁移越快。 b. Agarose concentration 琼脂糖浓度浓度越低，迁移越快。 c. Conformation of the DNA DNA的构象环状的或带切口环状的DNA通常比线状的DNA迁移要快。 d. Voltage per cm distance between electrodes 两个电