Experiment 4
Agarose Gel Electrophoresis
Electrophoresis is atechnique used to separate and sometimes purify macromolecules-especiallyproteins and nucleic acids, which differ in size, charge or conformation. Assuch, it is one of the most widely used techniques in biochemistry andmolecular biology. When charged molecules are placed in an electric field, theymigrate toward either the positive (anode) or negative (cathode) pole accordingto their charge. In contrast to proteins, which can have either a net positiveor net negative charge, nucleic acids have a consistent negative chargeimparted by their phosphate backbone, and migrate toward the anode.Macromolecules are electrophoresed within a matrix or"gel". According to the difference of materials used to make the gel,gel electrophoresis may be divided into two classes: agaroseand polyacrylamide electrophoresis. Agarose gel electrophoresis is now the most populartechnique used to separate and purify DNA fragments.
Purpose
By the end of this laboratory exercise, youshould be able to describe how to run an agarose gel.
Agarose is a polysaccharideextracted from seaweed. Agarose gels have alarge range of separation, but relatively low resolving power. Byv执业医师arying the concentration of agarose, fragments ofDNA from about 200 to 50,000 bp can be separatedusing standard electrophoretic techniques. The higherthe agarose concentration, the "stiffer"the gel. Higher concentrations of agarose facilite separation of small DNAs,while low agarose concentrations allow resolution oflarger DNAs. It is typically used at concentrationsof 0.5 to 2%. The distance DNA has migrated in the gel can be judged byvisually monitoring migration of the tracking dyes. Bromophenolblue and xylene cyanol dyesmigrate through agarose gels at roughly the same rateas double-stranded DNA fragments of 300 and 4000 bp,respectively. When adequate migration has occured, DNA fragments can be visualized by stainingwith ethidium bromide. This fluorescent dyeintercalates between bases of DNA and RNA. It is often incorporated into thegel so that staining occurs during electrophoresis, but the gel can also bestained after electrophoresis by soaking in a dilute solution of ethidium bromide. To visualize DNA or RNA, the gel isplaced on a ultraviolet transilluminator.
Reagentsand apparatus
ü Agarose.
ü TAE(Tris-Acetate,EDTA)[concentratedstocksolution 50X: Trisbase,242g; glacial acetic acid, 57.1ml; 0.5M EDTA (pH8.0),100 ml; add to 600mldH2O, stir vigorously, bring to 1000ml with dH2O].
ü Ethidium bromidestock (10 mg/ml): add 1 g of ethidium bromide to 100ml of H2O, stir with a magnetic stirrer for several hours and trasfer to adark bottle and store at 4℃(Ethidium bromide is a powerful mutagen.Wear gloves and a mask when weighting it out. In case of contact, immediatelyflush with copious amounts of water).
ü TBE [5x stock solution(1 liter): 54 g Tris base, 27.5 g boric acid, 20 ml 0.5 M EDTA, pH 8.0].
ü 10 x loading buffer: 0.25% bromophenolblue, 0.25% xylene cyanolFF, 15% Ficoll in water.
ü Equipment: beaker, graduated cylinder, stirbar, microwave, Pan balace, gel bed, comb,electrophoresis tank, and power supply.
Precedures
Preparation ofthe gel
1. Set upthe gel apparatus as demonstrated. The comb should be straight, and thereshould be a few millimeters of clearance between the bottom of the comb and thebottom of the gel tray.
2. Weigh propoeramount of agarose(e.g.,1.0g agarosein 100ml TAE buffer makes 1% agarose) and dissolve inTAE buffer by heating it in a microwave.
3. Pour the agaroseslowly (aviod bubbles) onto a gel bed with the combinserted. Use the plastic transfer pipette to remove the bubble if there isany. Currently, many varieties of gel beds are available commercially and thosecan be used according to the instruction. However, the “traditional”, as wellas economical and simple gel bed can be made by sealing a proper sized plasticor a glass with the masking tape. Make sure the seal is tight to prevent agarose from leaking, and this can be achieved by runningyour finger along the edge of the gel bed several times.
4. Let gel polymerize for about20 to 60 min depending upon the size of the gel.
5. While the agarose gel is polymerizing, prepare the DNA sample and mixproper amount of DNA with the loading buffer containing the dye such as bromophenolblue.
6. Remove the comb after the agarose gel has polymerized and place the gel bed onto theelectrophoresis tank with the wells near the cathode (black terminal). Fill thetank with proper amount of TAE buffer. Usually the buffer is about 1 cm abovethe gel. Add ethidium bromide (final concentration is1 μg/ml) to the TAE buffer and mixit well. Alternatively, ethidium bromide can be addedto the agarose gel. This can be done by boiling the agarose in the microwave and cooling to about 50℃ and then adding ethidium bromide.
LoadingDNA samples and gel running
7. Hold thepipette perpendicular to the well and add DNA sample slowly with the pipettetip just beneath the opening of the well.
8. After allthe samples have been loaded to the wells, connect the gel tank with the power supplyproperly(black to cathode[-] and red to anode[+]). Set the voltage and timebefore turning on the power supply.
9. Make surethe leads have been properlyconnected by watching the platinum wire at the black terminal near the wells.Bubbles should be slowly rising if the leadshave a good connection.
10. Allowelectrophoresis to progress for appropriate time. The timing of electrophoresisdepends on the length of the gel and the amount of voltage applied. The longerthe gel and the lower the voltage, the longer time is needed. However, highvoltages are significantly less effective at resolving large DNA fragments.
Gel Interpretation
Theuncut DNA lane may have several bands in it. This occurs because国家医学考试网 the migrationof plasmid DNA in an agarosegel depends on its molecular conformation as well as its size in base pairs. Plasmid DNA can exist in any one of three majorconformations:
Supercoiled -Although a plasmid is usually pictured as an opencircle, within a bacterial cell the DNA strand is coiled around histone-like proteins to form a compact structure. This iscalled supercoiling and this form of the plasmid will move the fastest through the gel due to itscompact structure.
Nicked -During plasmid DNA replication, topoisomeraseI 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 arelaxed open circular structure. This form is the slowest migrating form of plasmid. Its “floppy” molecular shape impedes movementthrough the agarose gel.
Linear -Linear plasmid DNA occurs when damage results instrand nicks directly opposite each other on the DNA helix. This DNA moves at arate intermediate between supercoiled and nicked plasmid DNA. The presence of linear DNA in a plasmid preparation is a sign of either nuclease contaminationor sloppy lab procedure.
Gelphotography
DNA fragments separated on the agarosegel after electrophoresis can be visualized and photographed under the UVlight. Many kinds of cameras are available commercially and they can be easilymanipulated following the instructions. In principle, the aperture and thespeed are the two key factors for taking a good picture. For example, toincrease exposure, i.e. to make image more intense, a longer exposure time isrequired and this can be achieved by slowing the shutter speed, or increasingthe aperture. In contrast, to decrease exposure, i.e. to make pictures darker,a shorter exposure time or smaller lens opening is required.
Notes
1. Both TAEand TBE are common buffer used. TBE has relative higher buffering capacity thanTAE.
2. Theloading dye bromophenol blue migrates with DNA about0.5 kb and provides an index of the mobility of the fastest fragments.
3. Themigration of the DNA depends on the following factors:
a) Molecularsize of DNA － thesmaller the DNA, the faster the migration.
b) Agarose concentration – the lower theconcentration, the faster the migration.
c) Conformationof the DNA – circular or nicked DNA often migrate faster: than that of linearDNA.
d) Voltageper cm distance between electrodes – the higher the voltage, the faster themigration.
4. Troubleshooting
If the DNA bands are not sharp and uniform,it may be due to the following reasons:
a) OverloadedDNA
b) Voltagetoo high
c) Torn well
d) Bubble ingel
5. Photographequipment maintenance
实验四 琼脂糖凝胶电泳
导言
电泳常用于分离有时也用于纯化那些分子大小、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。当带电分子置于电场中，根据它们所带电荷的不同，不是向正极（阳极)移动，就是向负极（阴极)移动。核酸与蛋白质不同，蛋白质分子可以带正电也可以带负电，而核酸却只能带负电，这是由其磷酸盐分子骨架所决定的，且只能向阳极泳动。生物大分子是在某种基质或“凝胶”中迁移的，根据制备凝胶所用材料的不同，凝胶电泳又分为两类：琼脂糖和聚丙烯酰胺凝胶电泳。琼脂糖凝胶电泳已成为分离和纯化DNA片断应用最普遍的技术。
目的
通过本实验掌握琼脂糖凝胶电泳的操作方法。
原理
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的分离，而较低浓度的琼脂糖胶则可以分离较大的DNA片断。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。溴酚蓝和二甲苯青染料在琼脂糖凝胶中的迁移速率大致分别与300和4000 bp大小的双链DNA片断相同。迁移足够距离后，就可以通过溴化乙锭染色来观察DNA片断。溴化乙锭是一种荧光染料，它嵌插在DNA和RNA碱基之间。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溴化乙锭溶液中进行染色。必须将凝胶置于紫外分析检测器中才可以对凝胶中的DNA或RNA进行观察。
试剂与器材
ü 琼脂糖
ü TAE（Tris-乙酸，EDTA)[50倍的浓缩储备液：Tris,242g;冰醋酸57.1ml;0.5M EDTA(pH8.0),100ml;加入600ml蒸馏水，剧烈振摇，再加入蒸馏水至1000ml。]
ü 溴化乙锭储备液（10mg/ml)：在100ml水中加入1g溴化乙锭，用磁搅拌器搅拌数小时，转移到黑色瓶中，4℃保存（溴化乙锭是强诱变剂，在称取时务必带上手套、面具。一旦接触到，要立即用大量的水冲洗。)
ü TBE[5倍储备液（1升)：54gTris碱，27.5g 硼酸，20ml 0.5M EDTA,pH8.0].
ü 10×加样缓冲液:0.25％溴酚蓝,0.25%二甲苯青FF，15％聚蔗糖水溶液。
ü 实验器材：广口烧杯，量筒，搅拌棒，微波炉，托盘天平，凝胶板，梳子，电泳槽，电源。
实验步骤
Ⅰ. 凝胶的制备
1.如图所示搭好凝胶电泳槽。梳齿应该保持笔直，在梳齿底部与胶槽之间应保持几毫米的间隙。
2.称取适量琼脂糖，加入Tris-乙酸缓冲液中，用微波炉加热至溶解。（例如称取1.0g琼脂糖，溶于100ml Tris-乙酸缓冲液中，制成1％的琼脂糖)
3. 将琼脂糖溶液缓缓倒入插有齿梳的凝胶板（避免产生气泡)。如果有气泡，用巴斯德吸管去除。目前，许多类型的凝胶板都可以购买得到，按照说明书操作使用即可。而传统的经济简便的方法是用胶带将合适大小的塑料板或玻璃板的边缘封住，形成一个胶模，以此作为凝胶板。可用手指在凝胶板边缘来回触摸数次，确证密封完全，以防琼脂糖泄漏。
4. 根据凝胶的大小，让其凝固大约20至60分钟。
5.在琼脂糖凝固过程中，准备好DNA样品，且向其中加入含有染料如溴酚蓝的加样缓冲液。
6.琼脂糖凝胶凝固之后，移走齿梳，将凝胶板移至电泳槽上，加样孔在靠近阴极的一端（黑色端)。向槽中加入适量的TAE缓冲液，通常应没过胶面1cm。再加入溴化乙锭，混合完全（其最终浓度达到1 μg/ml)。另一种方法是将溴化乙锭加入凝胶中：用微波炉将琼脂糖煮沸，然后冷却至50℃，加入溴化乙锭。
Ⅱ.加样、电泳
7. 用移液枪缓慢将DNA样品垂直加入加样孔直至其开口下方。
8. 加完所有样品后，将电泳槽与电源正确连接（黑色对阴极，红色对阳极)。打开电源之前要调好电压和时间。
9. 观察铂丝是否已连接到加样孔附近的黑色端以确保导线连接正确。如果导线连接正确，会有气泡缓慢上升。
10. 电泳过程需要一定的时间，其长短取决于凝胶的长度及使用的电压大小。凝胶越长电压越低，则所需时间就越长。但是，在分离较大的DNA片断时，使用高电压效果不好。
Ⅲ.凝胶（图象)解释
未切割质粒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可以下列三种主要构象中的任何一种形式存在：
超螺旋－尽管质粒通常以开环的形式进行描述，然而在细菌细胞内DNA链却是盘绕在组蛋白样的蛋白质周围形成一种致密的结构。这就是所谓超螺旋结构，由于其结构致密，它在凝胶中的泳动速度最快。
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最慢，其“松散”的分子形式阻碍了它在琼脂糖凝胶中的运动。
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的泳动速率介于超螺旋与切口质粒DNA之间。质粒制备过程中出现线性DNA说明存在核酸酶污染或实验操作有问题。
IV凝胶摄影
电泳之后，在紫外光下可观察到DNA片断在琼脂糖凝胶中得到分离，同时可对凝胶进行拍照。市面上可购买到各种照相机，在说明书的指导下，很容易操作。大体上，要拍好一张照片，照相机的光圈孔径和感光率是关键因素。例如，为了增加曝光度,使影象密度更高，需要加长曝光时间，这可通过减慢快门速度或增加感光率得以实现。与之相反，若要降低曝光度，使影象更模糊，则要缩短曝光时间或减小透镜孔径。
注意事项
1.TAE 和 TBE均为常用的缓冲液。TBE比TAE有相对高的缓冲能力。
2. 加样染料溴酚蓝可与长度约为0.5kb的DNA一起迁移，可用于指示迁移率最高的片断。
3. DNA的迁移速率取决于以下因素:
a)DNA的分子大小－分子量越小，迁移越快。
b)琼脂糖浓度－浓度越低，迁移越快。
c) 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)两个电极之间单位厘米的电压－电压越高，迁移越快。
4. 问题及原因
如果DNA条带不够窄且不够均匀，可能是由以下原因所引起:
a) DNA过载
b) 电压过高
c) 加样孔破损
d) 凝胶中有气泡
5. 摄影器材的保养
