2020/7/21,1,Agarose Gel Electrophoresis 瓊脂糖凝膠電泳,2020/7/21,2,After the experiment is finished, students should know how to make an agarose gel.,掌握瓊脂糖凝膠電泳的操作的方法。,一、實驗目的 (Experimental Purpose),2020/7/21,3,Gel electrophoresis,2020/7/21,4,二、實驗原理(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.,電泳常用于分離和純化那些分子大小、電荷性狀或分子構(gòu)象有所不同的生物大分子尤其是蛋白質(zhì)和核酸。正因為如此，電泳已成為生物化學和分子生物學中應用最為廣泛的技術(shù)之一。,2020/7/21,5,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.,瓊脂糖是一種海藻多糖，瓊脂糖膠分離范圍很大，但其分辨率卻相對較低。通過改變瓊脂糖凝膠的濃度，應用標準的電泳技術(shù)可以分離200到50,000 bp 大小的 DNA 片斷。,瓊脂糖凝膠濃度越大，凝膠就越硬。較高濃度的瓊脂糖膠有利于較小的DNA片斷分離，而較低濃度的瓊脂糖膠則可以分離較大的DNA片斷。,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.,2020/7/21,6,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.,瓊脂糖膠濃度一般在0.5到2之間。,通過觀察示蹤染料的遷移距離可以判斷DNA的遷移距離。溴酚藍染料在瓊脂糖凝膠的遷移速率最大。,當遷移足夠距離后，就可以通過Gelview染色來觀察DNA片斷。,When adequate migration has occured, DNA fragments can be visualized by staining with Gelview.,2020/7/21,7,2020/7/21,8,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是一種熒光染料。它可以在做膠時混入其中在電泳時進行染色，也可以待電泳完成后將凝膠浸泡在稀釋的Gelview 溶液中進行染色 。但必須將凝膠置于紫外透射儀中才可以對凝膠中的DNA或RNA進行觀察。,2020/7/21,9,三、試劑與器材（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,2020/7/21,10,. Preparation of the gel （凝膠的制備） 1.制備1瓊脂糖凝膠。稱取0.5g瓊脂糖，放人錐形瓶中，加入50mL的 0.5TBE 緩沖液，放入微波爐加熱至完全溶化，則為1瓊脂糖凝膠液。（由于蒸發(fā)作用，溶解前在容量瓶上作一個記號，溶解后用三蒸水補足）,四、實驗步驟(Experimental Procedures),2020/7/21,11,2.制膠器的安裝 取多功能制膠器，洗凈，晾干； 將多功能制膠器放置于一水平位置，選擇126cm制膠架，然后選擇1.5mm 18teeth的梳子（最大加樣量25l）； 將所選擇規(guī)格的梳子插入制膠架的定位槽中。,2020/7/21,12,3. 將熔化的瓊脂糖凝膠液轉(zhuǎn)入Gelview專用的三角瓶中，然后加入Gelview 5l。 4. 將冷到60左右的瓊脂糖凝膠液，緩緩倒入所選擇的制膠槽內(nèi)，直至有機玻璃板上形成一層均勻的膠面(注意不要形成氣泡)。 5. 待膠凝固后（30-60min），輕輕拔掉梳子，將凝膠盤從制膠槽中取出，放入電泳槽內(nèi)。 6. 加入電泳緩沖液(0.5)至電泳槽中。,2020/7/21,13,. Loading DNA samples (加樣),用移液槍緩慢將DNA樣品垂直加入加樣孔直至開口下方。 1.DNA samples ： 10l 2. Loading buffer (6X)：2l 3. DNA markers ：6l,Total 12l,2020/7/21,14,.Gel（電泳）,1. 接通電泳槽與電泳儀的電源(注意正負極，DNA片段從負極向正極移動)。保持電壓 6080 V。 2. 當溴酚藍染料移動到距凝膠前沿1-2cm處，停止電泳。,2020/7/21,15,. Gel Interpretation （凝膠圖像解釋）,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:,未切割的質(zhì)粒DNA在其泳道上也許會出現(xiàn)幾個條帶，之所以這樣是由于質(zhì)粒DNA在瓊脂糖凝膠中的遷移距離是由其分子構(gòu)象及其堿基對大小所決定的。質(zhì)粒DNA以下列三種主要構(gòu)象中的任何一種形式存在：,2020/7/21,16,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.,超螺旋質(zhì)粒在細菌細胞以超螺旋結(jié)構(gòu)存在，由于其結(jié)構(gòu)致密，它在凝膠中的泳動速度最快。,2020/7/21,17,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.,切口在質(zhì)粒DNA復制過程中，拓撲異構(gòu)酶I會在DNA雙螺旋中的一條鏈中引入一個切口，解開質(zhì)粒的超螺旋。在質(zhì)粒分離過程中由于物理剪切和酶的切割作用同樣也會在超螺旋質(zhì)粒中引入切口從而產(chǎn)生松散的開環(huán)結(jié)構(gòu)。這種形式的質(zhì)粒遷移速率介于超螺旋和線性DNA之間。,2020/7/21,18,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.,線性當DNA損傷在DNA雙鏈相對應的兩條鏈上同時產(chǎn)生切口時，就會出現(xiàn)線性質(zhì)粒DNA，這種DNA的泳動速率最慢，質(zhì)粒制備過程個出現(xiàn)線性DNA說明存在核酸酶污染或?qū)嶒灢僮饔袉栴}。,2020/7/21,19,五、實驗結(jié)果 (experimental results),1、DNA相對分子質(zhì)量標準物（maker） 2、pUC19質(zhì)粒DNA經(jīng)EcoRI完全酶解 3、 pUC19質(zhì)粒DNA經(jīng)EcoRI部分酶解 4、自提的pUC19質(zhì)粒DNA（此結(jié)果提得較好，為1條帶，以超螺旋為主。）,2020/7/21,20,六、注意事項(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，遷移越快。 b. Agarose concentration 瓊脂糖濃度濃度越低，遷移越快。 c. Conformation of the DNA DNA的構(gòu)象環(huán)狀的或帶切口環(huán)狀的DNA通常比線狀的DNA遷移要快。 d. Voltage per cm distance between electrodes 兩個電極之間單位厘米的電壓電壓越高，遷 移越快。,2020/7/21,21,Troubleshooting .（問題及原因） If the DNA bands are not sharp and uniform, it may be due to the following reasons 如果DNA條帶不夠窄且不夠均勻，可能是內(nèi)以下原因所引起： a. Overloaded DNA DNA過載 b. Voltage too high 電壓過高 c. Torn well 加樣孔破損 d. Bubble in gel 凝膠中有氣泡,