琼脂糖凝胶电泳Agarosegelelectrophoresis
琼脂糖凝胶电泳(Agarose gel electrophoresis)
You may also like... Restriction Digest of Plasmid DNA Purifying DNA from an Agarose Gel DNA Ligation This website uses cookies to ensure you get the best experience. By continuing to use this site, you agree to the use of cookies. Addgene is open and shipping orders.•Pending order? We will email to confirm that your organization can accept shipments.•Conducting coronavirus or COVID-19 research? Email us at help@ with your order or deposit number so we can prioritize it.Learn more about our current shipping status and COVID-19 resources .Agarose Gel Electrophoresis IntroductionGel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) forvisualization and purification. Electrophoresis uses an electrical field to move the negatively charged DNA throughan agarose gel matrix toward a positive electrode. Shorter DNA fragments migrate through the gel more quickly thanlonger ones. Thus, you can determine the approximate length of a DNA fragment by running it on an agarose gelalongside a DNA ladder (a collection of DNA fragments of known lengths).Last Update: Feb. 20, 2018EquipmentReagentsProcedurePouring a Standard 1% Agarose Gel:1.Measure 1 g of agarose.2.Mix agarose powder with 100 mL 1xTAE in a microwavable flask. See TAE Recipe .Note: Make sure to use the same buffer as the one in the gel box(do not mix different buffers and do not use water).3.Microwave for 1-3 min until the agarose is completely dissolved (but donot overboil the solution, as some of the buffer will evaporate and thusalter the final percentage of agarose in the gel. Many people prefer tomicrowave in pulses, swirling the flask occasionally as the solutionheats up.).CAUTION:HOT! Be careful stirring, eruptive boiling can occur.4.Close •Casting tray •Well combs •Voltage source •Gel box•UV light source •Microwave•TAE (recipe here )•Agarose•Ethidum bromide (stock concentration of 10 mg/mL)*Pro-Tip* Agarose gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to beseparated - see FAQs below . Simply adjust the mass of agarose in a given volume to make gels of other agaroseconcentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2% gel).*Pro-Tip* TBE can be used instead of TAE, labsusually use one or the other, but there is very littledifference between the two.Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins.5.(Optional) Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.CAUTION: EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical.Note: If you add EtBr to your gel, you will also want to add it to the running buffer when you run the gel. If you do not add EtBr to the gel and running buffer, you will need to soak the gel in EtBr solution and then rinse it in water before you can image the gel.6.Pour the agarose into a gel tray with the well comb in place.7.Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.Loading Samples and Running an Agarose Gel:1.Add loading buffer to each of your DNA samples.Note: Loading buffer serves two purposes: 1) it provides a visible dye thathelps with gel loading and allows you to gauge how far the DNA hasmigrated; 2) it contains a high percentage of glycerol that increases thedensity of your DNA sample causing it settle to the bottom of the gel well,instead of diffusing in the buffer.2.Once solidified, place the agarose gel into the gel box (electrophoresis unit).3.Fill gel box with 1xTAE (or TBE) until the gel is covered.4.Carefully load a molecular weight ladder into the first lane of the gel.Note: When loading the sample in the well, maintain positivepressure on the sample to prevent bubbles or buffer from enteringthe tip. Place the very top of the tip of the pipette into the buffer justabove the well. Very slowly and steadily, push the sample out andwatch as the sample fills the well. After all of the sample is unloaded,push the pipettor to the second stop and carefully raise the pipettestraight out of the buffer.5.Carefully load your samples into the additional wells of the gel.6.Run the gel at 80-150 V until the dye line is approximately 75-80% of theway down the gel. A typical run time is about 1-1.5 hours, depending onthe gel concentration and voltage.Note: Black is negative, red is positive. The DNA is negatively charged and will run towards the positive electrode. Always Run to Red.7.Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.8.(Optional) If you did not add EtBr to the gel and buffer, place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr,place on a rocker for 20-30 mins, replace EtBr solution with water and destain for 5 mins.ing any device that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to theirappearance on the gel.Note: When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a lab coat.Analyzing Your Gel:Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can infer the size of the DNA in your sample lanes. For more details on doing diagnostic digests and how to interpret them please see the Diagnostic Digest page.*Pro-Tip* It is a good idea to microwave for 30-45 sec, stop and swirl, and then continue towards a boil. Keep an eye on itthe solution has a tendancy to boil over. Placing saran wrap over the top of the flask can help with this, but is not necessaryif you pay close attention.*Pro-Tip* Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb ortowards the sides/edges of the gel with a pipette tip.*Pro-Tip* If you are in a hurry, the gel will set more quickly if you place the gel tray at 4 °C earlier so that it is already coldwhen the gel is poured into it.*Pro-Tip* Remember, if you added EtBr to your gel, add some to the buffer as well. EtBr is positively charged and will run theopposite direction from the DNA. So if you run the gel without EtBr in the buffer you will reach a point where the DNA will bein the bottom portion of the gel, but all of the EtBr will be in the top portion and your bands will be differentially intense. Ifthis happens, you can just soak the gel in EtBr solution and rinse with water to even out the staining after the gel has beenrun, just as you would if you had not added EtBr to the gel in the first place.*Pro-Tip* If you will be purifying the DNA for later use, use long-wavelength UV and expose for as little time as possible tominimize damage to the DNA.Purifying DNA from Your Gel:If you are conducting certain procedures, such as molecular cloning, you will need to purify the DNA away from the agarose gel. For instructions on how to do this, visit the Gel Purification page.Tips and FAQ•How do you get better resolution of bands?A few simple ways to increase the resolution (crispness) of your DNA bands include: a) running the gel at a lower voltage for a longer period oftime; b) using a wider/thinner gel comb; or c) loading less DNA into the well. Another method for visualizing very short DNA fragments ispolyacrylamide gel electrophoresis (PAGE), which is typically used to separate 5 - 500 bp fragments.•How do you get better separation of bands?If you have similarly sized bands that are running too close together, you can adjust the agarose percentage of the gel to get better separation. A higher percentage agarose gel will help resolve smaller bands from each other, and a lower percentage gel will help separate larger bands.•10% Rule:For each sample you want to load on a gel, make 10% more volume than needed because several microliters can be lost in pipetting. Forexample, if you want to load 1.0 μg in 10 μL, make 1.1 μg in 11 μL.Reference Page | Top | Index。
2琼脂糖凝胶电泳
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。
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：
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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.
When adequate migration has occured, DNA fragments can be visualized by staining with Gelview.
当迁移足够距离后，就可以通过Gelview染色来观察DNA片断。
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琼脂糖 0.3% 0.6% 0.9% 1.2% 1.5% 2%
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。
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五、实验结果 (experimental results)
琼脂糖凝胶电泳的理论技术和应用
琼脂糖凝胶电泳的理论技术和应用1 引言琼脂糖凝胶电泳（agarose gel electrophoresis）主要是应用琼脂糖凝胶作为支持物的电泳法，借助琼脂凝胶的分子筛作用，核酸片段因其分子量或者分子形状不同，电泳移动速度有差异而分离，这种技术时基因操作中常用的一种方法。
下面简单介绍琼脂糖凝胶电泳的理论技术及其应用。
2 琼脂糖凝胶电泳技术及其原理(1)电泳主要是指混悬于溶液中的样品电荷颗粒，在电场影响下向着与自身相反电荷的电极移动的现象，电泳技术是一种非常先进的检测手段，电泳技术与其它先进的技术相配合能够创造出非常之高的成果，这种技术能够使人们以最小的代价获得最大的利益。
电泳技术现在主要用于纯化以及分离DNA片段的一种最常用的技术。
原理：电泳是现在用于纯化以及分离DNA片段的最常用的技术，如果装备一块“胶”包含电解质的多孔支持介质，并把这些介质放置在静电场中，DNA分子将会随着向阳极移动，这主要是因为DNA分子沿着双螺旋骨架两侧带有含有电荷的磷酸根残基，当DNA的长度增加后，来自电场的驱动力以及凝胶的阻力之间的比率就会降低，并且不同长度的DNA片段就会出现不同的迁移率，所以就可以根据DNA分子大小使其分离。
这个过程可能是通过把分子量标准参照物或者是示踪燃料以及样品一起进行电泳检测分子量标准参照物也可以提供一个用于确定DNA片段大小的标准。
(2)琼脂糖凝胶电泳主要是采用琼脂糖作为支持介质的电泳方法，琼脂糖凝胶电泳技术的分析原理与其他支持物电泳的最主要的区别是它具有“电泳”和“分子筛”双重作用。
琼脂糖凝胶是一种网络结构，物质分子通过时会受到阻力，其中较大的分子物质在涌动时受到的阻力比较大，所以在凝胶中，带电颗粒的分离不仅与静电荷的性质以及数量有关，而且与废纸的大小有很大的关系，这就可以大大的提高了粪便能力，但是由于其孔径相差比较大，对于大多数蛋白质来说其分子筛效应很小，目前琼脂糖凝胶技术主要用于核酸的研究中。
Agarose gel electrophoresis(琼脂糖凝胶电泳)
Agarose gel electrophoresis（琼脂糖凝胶电泳）Agarose gel electrophoresis (2) is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be eluted from the gel. Prior to gel casting, dried agarose is dissolved in buffer by heating and the warm gel solution then is poured into a mold (made by wrapping clear tape around and extending above the edges of an 18 cm X 18 cm glass plate), which is fitted with a well-forming comb.The percentage of agarose in the gel varied. Although 0.7% agarose gels typically are used, in cases where the accurate size fractionation of DNA molecules smaller than 1 kb is required, a 1, 1.5, or 2% agarose gel is prepared, depending on the expected size(s) of the fragment(s). Ethidium bromide is included in the gel matrix to enable fluorescent visualization of the DNA fragments under UV light. Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. The DNA samples are mixed with gel tracking dye and loaded into the sample wells. Electrophoresis usually is at 150 - 200 mA for 0.5-1 hour at room temperature, depending on the desired separation.When low-melting agarose is used for preparative agarose gels, electrophoresis is at 100-120 mA for 0.5-1 hour, again depending on the desired separation, and a fan is positioned such that the heat generated is rapidly dissipated. Size markers areco-electrophoresed with DNA samples, when appropriate for fragment size determination. Two size markers are used, phi-X 174 cleaved with restriction endonuclease HaeIII to identify fragments between 0.3-2 kb and lambda phage cleaved with restriction endonuclease HindIII to identify fragments between 2-23 kb. After electrophoresis, the gel is placed on a UV light box and a picture of the fluorescent ethidium bromide-stained DNA separation pattern is taken with a Polaroid camera.Protocol1. Prepare an agarose gel, according to recipes listed below, by combining the agarose (low gel temperature agarose may also be used) and water in a 500 ml Ehrlenmeyer flask, and heating in a microwave for 2-4 minutes until the agarose is dissolved.Genetic technology grade (800669) or low gel temperature (800259) agarose from Schwarz/Mann Biotech.2. Add 20X TAE and ethidium bromide (EtBr), swirl to mix, and pour the gel onto a taped plate with casting combs in place. Allow 20-30 minutes for solidification.3. Carefully remove the tape and the gel casting combs and place the gel in a horizontal electrophoresis apparatus. Add 1X TAE electrophoresis buffer to the reservoirs until the buffer just covers the agarose gel.4. Add at least one-tenth volume of 10X agarose gel loading dye to each DNA sample, mix, and load into the wells. Electrophorese the gel at 150-200 mA until the required separation has been achieved, usually 0.5-1 hour (100-120 mA for low gel temperature agarose), and cool the gel during electrophoresis with a fan. Visualize the DNA fragments on a long wave UV light box and photograph with a Polaroid camera。
Agarose_Gel_Electrophoresis
Agarose Gel Electrophoresis琼脂糖凝胶电泳三、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].实验室：TAE50×3. 10 × loading buffer: 0.25% bromophenol blue, 40% sucrose in water.实验室：碧云天 6×4. Equipment: beaker, graduated cylinder, stir bar, microwave, Pan balance, comb, electrophoresis tank, and Electro-phoresis System , Ultraviolet transilluminator.5. EB实验室：分装5mg/mL四、实验步骤(Experimental Procedures)Ⅰ. 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 1×TAE 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或称量质量，溶解后用三蒸水补足）2.制胶器的安装①取多功能制胶器，洗净，晾干；②将多功能制胶器放置于一水平位置，选择12×6cm制胶架，然后选择1.5mm 18teeth的梳子（最大加样量25µl）；③加入电泳缓冲液(1×)至电泳槽中3. 将熔化的琼脂糖凝胶液转入三角瓶中，然后加入EB 5µl。
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)。
五种电泳技术的比较
五种电泳技术的比较SDSPAGE名词解释：相对迁移率（Rf）问答题:1.简述SDS-PAGE的基本原理。
2.影响SDS电泳的关键因素有哪些？AGE名词解释1.迁移率2.电渗3.电泳问答题1.影响电泳迁移率的因素有哪些？2.试述琼脂糖凝胶电泳分离脂蛋白的原理。
CAME1.CAME的基本原理是什么？2.CAME分离血清蛋白电泳时应注意哪些问题？PAGE名词解释1.凝胶总浓度2.交联度问答题1.与CAME相比，PAGE有哪些特点。
2.试比较CAME与PAGE操作的区别。
3.简述不连续PAGE的原理。
1.琼脂糖凝胶电泳Agarose Gel ElectrophoresisGel Electrophoresis ：由琼脂、琼脂糖、淀粉胶及聚丙烯酰胺等物质作支持体的电泳。
特点（characteristic）：1.可以制成非常均匀的凝胶，带电质点在凝胶的孔中泳动。
2. 电泳操作方法简便，电泳速度快。
3. 分辨率高，重复性好，电泳图谱清晰。
4. 适用于生化，免疫等定性定量测定。
（一）优点（advantage)1.因不含硫酸根和羧基，几乎消除了琼脂的电渗。
2.对蛋白质吸附极微，故无拖尾现象。
3.凝胶结构均匀，孔径较大，可用来分离酶的复合物、核酸、病毒等大分子物质。
4.透明度较好，可直接或干燥成薄膜后进行染色。
5.不吸收紫外光，可直接利用紫外光吸收法作定量测定。
6.有热可逆性。
（二）缺点(disadvantage)1.机械强度差，易破碎，浓度不能太低。
2.易被细菌污染，不易保存，临用前配制。
3.琼脂糖支持层上的区带易于扩散，电泳后必须立即固定染色。
4.与PAGE相比，分子筛(molecular sieve)作用小，区带少。
应用1. 适用于大分子的核酸、核蛋白等的分离、鉴定及纯化2. 临床生化检验中常用于LDH、CK等同工酶的分离与检测3. 为不同类型的高脂蛋白血症、冠心病等提供生化指标影响迁移的因素the size of the moleculeconformation of the moleculethe agarose concentration of a gelVoltage百分浓度和分辨率限制Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer.Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel 聚丙烯酰胺is more appropriate in this case.Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1% gels are common for many applications.琼脂糖凝胶分离血浆脂蛋白原理：血清脂蛋白经饱和苏丹黑B预染后，以琼脂糖凝胶为支持介质，在pH8.6巴比妥缓冲液中电泳，根据各脂蛋白的组成、大小、形状分离成不同区带。
琼脂糖凝胶电泳法（agarosegelelectrophoresis）
琼脂糖凝胶电泳法（agarose gel electrophoresis）agarose gel electrophoresisThe principle of 1. agarose gel electrophoresisAgarose gel electrophoresis is a standard method for isolation, identification and purification of DNA fragments. The technique is simple and rapid and can be used to distinguish DNA fragments that cannot be isolated by other methods such as density gradient centrifugation. When the dye Ethidium (bromide, EB) is dyed with low concentration fluorescent dye, DNA bands of 1-10ng can be detected at least under ultraviolet light so that the position of DNA fragments in the gel can be determined. In addition, a specific DNA band can be recovered from the electrophoretic gel for subsequent cloning operations.Agarose can be made into various shapes, sizes, and porosities. Agarose gel separated DNA slices with a wide range of sizes. Agarose gels of different concentrations could separate DNA fragments from 200bp to near 50kb. Agarose is commonly used in horizontal devices for electrophoresis under constant electric field with constant intensity and direction.Agarose is mainly used as a solid support substrate in DNA electrophoresis, and its density depends on the concentration of agarose. In the electric field, the negative charge DNA moves towards the anode at the neutral pH, and the migration rate is determined by the following factors:Molecular size of 1. DNA:A linear double stranded DNA molecules in a certain concentration in agarose gel migration rate and molecular weight of DNA is inversely proportional to the logarithm of the molecular, the greater the resistance is greater, more difficult in the gel pores. Therefore, the slower migration.2. agarose concentrationA linear DNA molecule of a given size has a different migration rate at different concentrations of agarose gels. The logarithm of mobility of DNA electrophoresis is linearly related to gel concentration. The choice of gel concentration depends on the size of the DNA molecule. The concentration required for separating DNA fragments less than 0.5kb is 1.2-1.5%, and the concentration of DNA molecules larger than 10KB is 0.3-0.7%, and the concentration of DNA fragments between them is0.8-1.0%.Conformation of 3.DNA moleculeWhen the DNA molecule is in different conformation, it moves in the electric field, not only in relation to the molecular weight, but also to its conformation. The same molecular weight of the linear, open loop and ultra helical DNA in agarose gel moving speed is not the same, ultra helical DNA moving fastest, and linear double stranded DNA move the slowest. As in the electrophoresis of plasmid purity found several gel DNA with plasmid DNA is difficult to determine the cause of different conformation or because of containing other caused by DNA, can be gradually recovered from the agarose gel, DNA, respectively,hydrolysis, using the same restriction enzyme and gel electrophoresis, as in the DNA appear on the map is the same. For the same kind of DNA.4 、 supply voltageAt low voltage, the migration rate of the linear DNA fragment is proportional to the applied voltage. But with the increase of field strength, the migration of DNA with different molecular weight fragment rate will increase with different amplitude, fragment is larger, because the field strength increases caused by migration rate increased more significantly, so the voltage increases, the effective separation range of agarose gel will be reduced. To maximize the resolution of the DNA fragment greater than 2KB, the voltage shall not exceed 5v/cm.5, the presence of embedded dyesEthidium bromide, a fluorescent dye, was used to detect DNA in agarose gels,The dye is embedded between the stacked base pairs, stretching the elongated and notched ring DNA, making it more rigid and reducing the linear DNA mobility by 15%.6. effect of ionic strengthThe composition and ionic strength of electrophoretic buffer affect the electrophoretic mobility of DNA. In the absence of ion existence (such as the misuse of distilled water gel), theconductivity minimum, DNA almost does not move in high ionic strength buffer in (such as the error plus 10 x buffer), has very high conductance and obvious heat production would cause serious gel melting or denaturation of DNA.For natural double stranded DNA, several commonly used electrophoretic buffers are TAE[, EDTA (pH8.0) and Tris- acetic acid], TBE (Tris-, boric acid and EDTA), TPE (Tris-, phosphoric acid and EDTA) are generally formulated as concentrated mother liquor and stored at room temperature.2. steps of agarose gel electrophoresis1. take 5 * TBE buffer, 20ml add water to 200ml, prepare 0.5 * TBE dilution buffer.2. glue preparation: weigh 0.4g agarose, a 200ml conical flask, adding 0.5 50ml * TBE dilution buffer, placed in a microwave oven (or electric heating) to remove all agarose melting, shake, this is the 0.8% agarose gel. During heating, the agarose particles attached to the bottle wall shall be shaken from time to time to enter the solution. Heating should be covered with sealing film to reduce moisture evaporation.3., the preparation of rubber board: plexiglass trough trough at the ends of each with rubber paste (width 1cm) sealed tightly. Place the sealed glue trough on the horizontal support, insert the sample comb, and pay attention to observe the gap between the comb tooth edge and the bottom of the rubber tank to keep the clearance of about 1mm. Adding to the ethidium bromide agarose liquid cooled to 45-60 DEG C in (EB) solution to thefinal concentration of 0.5 g /ml (or not added EB gel after electrophoresis, but EB solution with 0.5 g/ml staining). Absorb a small amount of molten agarose gel with a pipette and seal the inner side of the adhesive. After the agarose solution is coagulated, the remaining agarose is carefully poured into the gel groove to form a uniform adhesive layer. Pour glue when the temperature can not be too low, otherwise the solidification is uneven, the speed can not be too fast, otherwise prone to bubbles. After the gel has been completely solidified, dial the comb and pay attention not to damage the gel at the bottom of the comb. Then add 0.5 * TBE dilution buffer to the surface of the comb, just above the surface of the rubber sheet. Because of the edge effect, there will be some uplift near the sample slot, which prevents the buffer from entering the sample tank, so make sure the sample tank is filled with buffer.4. add sample: take 10 L enzymolysis liquid and mix with 2 l 6 * sample liquid, carefully add into the sample trough with micro liquid gun. If the content of DNA is low, the amount of sample can be increased according to the above proportion, but the total volume can not exceed the capacity of the sample tank. When each sample is added, replace the tip head to prevent contamination. Attention should be paid to handling the sample, avoiding damage to the gel or piercing the gel at the bottom of the sample slot.5. electrophoresis: after finishing the sample, close the electrophoresis cover and turn on the power immediately. The control voltage is kept at 60-100V and the current is above 40mA. When the br blue indicator band is moved to the gum 3/4, theelectrophoresis is stopped at all times.6. dyeing: no EB rubber plate, after electrophoresis, moved into 0.5 g/ml EB solution, dyeing at room temperature for 20-25 minutes.SevenObserve and photograph: adjust the shooting range and focal length of the lens under the long wavelength ultraviolet lamp with the wavelength of 254nm, and observe or dye the electrophoresis gel board with EB added. The presence of DNA showed a reddish orange fluorescence band visible to the naked eye. Finally, print the photos and make the relevant analysis records.Note: 1. observation of DNA can not be separated from ultraviolet transmittance instrument, but ultraviolet light has a cutting effect on DNA molecule. When the DNA is recovered from the glue, the illumination time should be shortened and the long wavelength ultraviolet lamp (300-360nm) should be adopted to reduce the ultraviolet ray cutting DNA.2.EB is a potent mutagen that has potential carcinogenicity and is moderately toxic. Therefore, gloves must be used when preparing and using it, and do not spill EB on the table or on the ground. Any container or article contaminated with EB must be specially treated before it can be washed or discarded.3. when the EB is too much, the gel is too dark and the DNA band is not clear, the gel can be distilled into distilled water andbe re observed after 30 minutes.4. avoid the formation of bubbles when preparing agarose gel5., the sample should be of appropriate concentration and smaller volume, with a micro syringe slowly add samples, point sample, the power must be in a closed state. In order to indicate the distance between electrophoresis and to prevent the sample from floating in the buffer of the electrophoresis bath, the sample buffer containing sucrose or glycerol and indicator (bromo phenol blue, xylene blue, etc.) is often added into the prepared sample. In addition, the salt content in the sample can not be too high, otherwise, the electrophoresis will occur in the disappearance of zones and the uneven front. The content of DNA in the sample should be no less than 0.1 mu g in each zone, and the concentration of DNA is too high, which will widen the electrophoresis zone and change the swimming distance of DNA.6., under the violet light observation, should wear protective glasses or plexiglass mask, so as not to damage the eye.。
血清蛋白(serumprotein)琼脂糖凝胶电泳
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2、制备琼脂糖凝胶板将已配制好的 0.5%琼 脂糖凝胶于沸水浴中加热融化，用吸管吸取凝胶 溶液浇注在载玻片上，约 3mL。静置半小时后凝 固。
3、点样将剪好的滤纸条对折，以折边在距 凝胶一端 2cm 处切一点样口。将毛细管插入预染 好的血清中，待吸入部分血清后，取毛细管使其
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【注意事项】
1、电泳样品应为新鲜的空腹血清。
2、加热溶化琼脂时，须防止水份蒸发过多。 琼脂糖凝胶最好随用随制，以免凝胶表面干燥， 影响分离效果。
琼脂糖电泳法测定多糖的操作规程
琼脂糖凝胶电泳法测定多糖的操作规程Protocol of agarose gel electrophoresis for analyzing polysaccharide实验仪器与材料：实验仪器：DYCp－3/C型电泳槽（Electrophoresis chamber）、制胶槽（gel form）和梳子（comb, 梳齿长1cm）北京市六一仪器厂（尺寸：11cmX6.5cm）（最大电压600v, 最大电流200mA）DYY-5型稳压稳流电泳仪(Electrophoresis power supply)北京市六一仪器厂STS-2型脱色摇床上海琪特分析仪器有限公司电子天平上海精科天平 FA2004320S pH计上海梅特勒－托利多公司密封式恒温可调电加热器浙江嘉兴市凤桥电热器厂微量加样器 (pipette) Gilson公司与微量加样器适配的吸头(tip) 购自天津联星生物公司材料:1 琼脂糖（Agarose）北京奥博星生物技术责任有限公司 BIOWEST AGAROSE (Regular ) Distributed by Shanghai Yito Enterprise Company Ltd. BR2 巴比妥钠（ Sodium barbital）中国医药（集团）上海试剂公司 CP3 乙二胺（配制电泳缓冲液用，本实验未选用）4 甲苯胺蓝（Toluidine Blue, Dimethyl toluthionine chloride）UNI-chem Chemical reagent (购自天津联星生物公司) CP5 冰醋酸（Acetic acid glacial）天津科威公司AR6 无水乙醇（Ethanol absolute）天津科威公司 AR7 十六烷基三甲基溴化铵(Cetyltrimethylammonium bromide)天津市远航化学品有限公司经销分析专用8溴酚蓝（Bromophenol Blue）购自天津联星生物公司ACS Grade9 甲酚红（指示剂，选用，本实验室未采用）10 标准硫酸化葡聚糖Dextran Sulphate或标准化肝素 Heparin（用于标定琼脂糖电泳凝胶，选用，本实验室未采用）11盐酸 (Hydrochloric acid)天津市大茂化学试剂厂12 爱茜蓝（Alcian Blue）染色剂13 多糖由本实验室自行制备14 乙酸钠(Sodium acetate anhydrous) 天津科威公司 AR (分子量82.03)15 胶带Masking tape, if needed to seal gel form16 托盘（尺寸）17 水（去离子水，过0.22μ滤膜处理）18 烧杯、量桶等玻璃容器若干19 记号笔实验准备1 按配方A配制巴比妥溶液500mL；2 按配方B配制染色液250mL;3按配方C配制脱色液500mL；4 按配方D配制琼脂糖溶液15mL;5 按配方F配制CTAB(十六烷基三甲基溴化铵溶液)(0.1%)100mL;6 按配方G配制醋酸钠缓冲液500mL；5 配制样品溶液（按照具体实验要求配制不同浓度的多糖溶液）；6 与相关实验人员协调，确定实验时间;7 检查电泳装置能否正常工作。
琼脂糖凝胶电泳英文
琼脂糖凝胶电泳英文Title: An Introduction to Agarose Gel ElectrophoresisAgarose gel electrophoresis is a widely used technique in molecular biology and biochemistry to separate and analyze DNA fragments or proteins based on their size. In this article, we will provide a brief overview of agarose gel electrophoresis, its principles, and its application in scientific research.## IntroductionAgarose gel electrophoresis is a powerful tool for the separation and analysis of nucleic acids and proteins. It involves the movement of charged molecules through a gel matrix in response to an electric field. The separation is based on the differences in the size and charge of the molecules present in the sample.## PrincipleAgarose is a polysaccharide derived from seaweed and is commonly used to prepare the gel matrix for electrophoresis. The gel is cast by mixing agarose powder with a buffer solution and heating it until the agarose is completely dissolved. Once the gel has solidified, it forms a matrix of pores through which molecules can migrate.When an electric current is applied to the gel, negatively charged molecules, such as DNA or proteins, migrate towards the positive electrode (anode), while positively charged molecules move towards the negative electrode (cathode). The migration rate is influenced by the size and charge of the molecules. Smaller molecules can pass through the pores more easily and migrate faster than larger molecules.## ProcedureAgarose gel electrophoresis involves several steps. Here is a general overview of the procedure:1. Prepare the gel: Dissolve agarose powder in a buffer solution by heating and cooling. Pour the liquid gel into a gel tray and insert a comb to create wells for sample loading.2. Prepare the samples: Mix the nucleic acid or protein samples with a loading buffer, which contains tracking dyes to visually monitor the progress of the electrophoresis.3. Load the samples: Carefully pipette the prepared samples into the wells of the gel.4. Run electrophoresis: Place the gel tray into an electrophoresis chamber filled with buffer solution. Connect the electrodes to the power supply and apply a voltage. The samples will start migrating through the gel.5. Visualize the results: After a suitable running time, turn off the power and carefully remove the gel tray. Stain the gel with dyes specific to the molecules being analyzed. The stained molecules can be visualized under UV light or using other detection methods.6. Analysis: Photograph or document the gel image to record the results. Analyze the migration pattern of the molecules based on their size and position on the gel.## ApplicationsAgarose gel electrophoresis finds numerous applications in scientific research, such as:1. DNA fragment analysis: Determining the size and quantity of DNA fragments produced by enzymatic digestion or PCR amplification.2. Protein analysis: Separating proteins based on size for identification or quantification.3. DNA sequencing: Separating and visualizing DNA fragments during the Sanger sequencing method.4. DNA fingerprinting: Analyzing a person's unique DNA profile by comparing the migration patterns of specific DNA markers.5. RNA analysis: Assessing the integrity and quantity of RNA molecules extracted from samples.Overall, agarose gel electrophoresis is an essential technique in molecular biology and provides valuable information about the size, quantity, and purity of nucleic acids and proteins.In conclusion, agarose gel electrophoresis is a versatile and widely used technique in the field of molecular biology. By understanding its principles and following the standard procedures, researchers can effectively separate and analyze DNA fragments or proteins, furthering our understanding of genetics and biochemistry.。
琼脂糖凝胶电泳详细过程与步骤-凝胶电泳加样
4）在实验加样中不一定每一个样品都换一个枪头，可在阳极 槽中反复吸打电泳缓冲液以清洗。（对于Southern印迹转 移和需要回收DNA片段的电泳，则应该每一个样品用一个 枪头加样，避免样品交叉污染。）
5）凝胶中加入EB进行电泳，便于紫外线下观察电泳状态， 但EB会导致线形DNA迁移率下降，这在酶切质粒与空白 对照质粒一起电泳时应值得注意。
缺点 1. 机械强度差，易破碎，浓度不能太低。 2. 易被细菌污染，不易保存，临用前配制。 3. 琼脂糖支持层上的区带易于扩散，电泳后必须
立即固定染色。 4. 与PAGE相比，分子筛作用小，区带少。
一、实验目的
掌握琼脂糖凝胶电泳分离DNA的原理和方法
二、实验原理
琼脂糖是一种天然聚合长链状分子，可以形成 具有刚性的滤孔，凝胶孔径的大小决定。
6）电泳过程中，溴化乙锭向负极移动，与DNA泳动的方向 相反，较长时间的电泳会造成靠正极方向的凝胶中溴化乙 锭含量低，会对含量较少的小分子DNA片段检测困难。处 理方法是：将凝胶在0.5ug/ml的EB溶液中染色30－45分 钟。
7 ） 判 断 正 负 极 的 另 一 方 法 是 负 极 气 泡 （ H2 ） 比 正 极 气 泡 （O2）多一倍。
低黏性低熔点琼脂 糖
凝结温度/℃ 35~38 40~42 34~43 25~35 35 8~15 25~30 38 30
熔化温度/ ℃ 90~95
85~90
85~95 63~65
65 40~45
70
不同厂家生 产的不同商 品其凝结温 度和熔化温 度有一定差 异
85
75
不同类型琼脂糖分离DNA片段的范围
sharpetal.1973 琼脂糖凝胶电泳
琼脂糖凝胶电泳（Agarose Gel Electrophoresis）是一种常见的生物分子分离技术，广泛应用于分离DNA、RNA和蛋白质等生物大分子。
它是静电力和大小分子筛分作用的结合体，可以在不破坏生物大分子结构的情况下对其进行分离和检测。
sharpetal.1973是一个常用的琼脂糖品牌，以下将介绍琼脂糖凝胶电泳的基本原理和实验步骤，并结合使用sharpetal.1973琼脂糖进行技术操作的一般经验。
一、琼脂糖凝胶电泳的基本原理1.电泳原理琼脂糖凝胶电泳是利用电场力与分子大小和电荷之间的相互作用来分离生物大分子的一种技术。
当样品加载在含有琼脂糖的凝胶糊中后，施加电场，分子在电场作用下向电极方向迁移，不同大小和电荷的分子会以不同速度迁移，从而实现分离。
2.琼脂糖的作用琼脂糖是一种多糖类物质，具有良好的水溶性和凝胶性，通常用于制备凝胶糊。
当琼脂糖和试剂混合后，可以制备成不同浓度的琼脂糖凝胶，用于分离不同大小的生物大分子。
琼脂糖凝胶的孔隙大小是影响分子迁移速度的重要因素。
二、琼脂糖凝胶电泳的实验步骤1. 凝胶制备a) 在无菌条件下，称取适量的琼脂糖粉末，加入缓冲液中，搅拌均匀；b) 将琼脂糖-缓冲液混合液置于微波炉中加热，使其充分溶解；c) 待液体冷却至约60摄氏度时，将其倒入凝胶板中，放置平整成型；d) 待凝胶固化后，将其置于电泳槽中，加入足够的缓冲液。
2. 样品处理a) 取适量的生物大分子试样，加入缓冲液和DNA标记物，混合均匀；b) 将混合液进行热变性处理，使DNA完全解链；c) 冷却后样品即可用于电泳。
3. 电泳操作a) 将待测样品注入凝胶槽中的样品孔中，注意不要产生气泡；b) 通电进行电泳，根据生物大分子的迁移速度和预设的电泳时间进行调整。
4. 结果分析a) 关闭电源后，取出凝胶板，进行染色和可视化；b) 观察凝胶上的分离明显程度和带形，分析样品中的DNA、RNA或蛋白质等情况。
三、sharpetal.1973琼脂糖的使用经验sharpetal.1973是一种常见的琼脂糖产品，广泛应用于生物分子分离和检测。
琼脂糖凝胶电泳(英文)
02
Operation process of agarose gel
electrophoresis experience
Experimental preparation
Preparation of reagents
Ensure all reagents are fresh and of high purity Prepare buffers according to the required pH and concentration
experience
目录
• Improvement and Development of Agarose Gel Electrophoresis Experiment
• Case analysis of agarose gel electrophoresis experience
01
Introduction of agarose gel
Equipment setup
Set up the electrophoresis chamber, power supply, and cooling system Check that the electrodes and clamps are in good conditions
Sample collection
03
Precautions for agarose gel
electrophoresis experience
Safety precautions
要点一
Wear protective clothing
Wear lab coat, gloves, and safety glasses to protect yourself from exposure to chemicals and biological materials
琼脂糖凝胶电泳
（四）琼脂糖凝胶电泳装臵
琼脂糖凝胶电泳的观察
在琼脂糖凝胶中加入溴化乙锭（ethidium bromide,
EB）或Golden view 。在紫外光下就可观察到明显DNA
的荧光谱带。
（五）琼脂糖凝胶电泳的基本步骤 和注意事项 1、制胶 2、上样 3、观察 4、拍照
EB — Carcinogen Ultra Violet— hurt your eyes
In both of these photos, a gel which has completely polymerized is now ready to use. Notice the spaces (called "wells") left by the removal of the comb. This can be seen better in the second photo. Both gels are resting on the glass support. The higher the agarose percentage, the bluer the gel.
The solvent used to dissolve the agarose. Rather than just use water, we use buffered solutions which allow the DNA to run smoothly through the gel. These solutions optimize the pH and ion concentration of the gel and will also bathe the gel as it is subjected to the electric current which actually moves the DNA through the gel.
DNA凝胶电泳
准备样品- DNA样品上样缓冲液混匀
迁移
紫外灯下观察电泳结果
DNA存在处显示出肉眼可辨的桔红色荧光条带
实验注意事项
EB是强诱变剂并有中等毒性，配制和使用时都应戴手套，
并且不要把EB洒到桌面或地面上。凡是沾污了EB的容器 或物品必须经专门处理后才能清洗。沾染了EB的垃圾要 专门处理后才可丢弃。
状DNA>开环DNA
琼脂糖凝胶中DNA的观察
溴化乙锭（EB）—DNA嵌合物：紫外光下发红色荧光
实验材料和试剂
实验材料：PCR产物 实验试剂：
① 5×TBE电泳缓冲液： ② 6×电泳上样缓冲液：0.25％ 溴粉蓝，40％(w/v) 蔗糖水溶 液，贮存于 4℃。 ③ 溴化乙锭(EB)溶液：5µ g/mL，避光储于室温即可。
dnadnaagarosegelelectrophoresis琼脂糖凝胶电泳是常用的用于分离鉴定dnarna分子混合物的方法这种电泳方法以琼脂凝胶作为支持物利用dna分子在泳动时的电荷效应和分子筛效应达到分离混合物的目的
实验六
DNA凝胶电泳
DNA agarose gel electrophoresis
实验原理（用于实验报告）
琼脂糖凝胶电泳是常用的用于分离、鉴定DNA、RNA分子混合物的方 法，这种电泳方法以琼脂凝胶作为支持物，利用DNA分子在泳动时的电荷 效应和分子筛效应，达到分离混合物的目的。DNA分子在高于其等电点的 溶液中带负电，在电场中向阳极移动。在一定的电场强度下，DNA分子的 迁移速度取决于分子筛效应，即分子本身的大小和构型是主要的影响因 素。DNA分子的迁移速度与其相对分子量成反比。 不同构型的DNA分子的迁移速度不同。如环形DNA分子样品，其中有 三种构型的分子：共价闭合环状的超螺旋分子（cccDNA）、开环分子 (ocDNA)、和线形DNA分子(IDNA)。这三种不同构型分子进行电泳时的迁 移速度大小顺序为:cccDNA＞IDNA＞ocDNA 当用低浓度的荧光嵌入染料溴化乙啶(Ethidium bromide, EB)染色， 在紫外光下至少可以检出1-10ng的DNA条带，从而可以确定DNA片段在凝 胶中的位置。此外，还可以从电泳后的凝胶中回收特定的DNA条带，用于 以后的克隆操作。
琼脂糖凝胶电泳(英文)
Notes
1. EB is a strong mutagen with moderately toxic, we
must wear disposable gloves in the operation to
prevent contamination.
2. When we use the gel imaging system, clean the
2
3
4
5
Experimental purposes
To master the basic principle and operating methods of agarose gel
electrophoresis
Experimental principle
1. Use of agarose gel electrophoresis
(2) Weighed amount of agarose, put it into triangular
flask, and pour the appropriate volume of 1×TAE,
then fully dissolve the agarose in the microwave oven.
evenly pour it into glue tank, then put the plastic tank at the level of
the table until completely cooled. (4) Remove the baffles on both sides, put the plastic tank into the electrophoresis tank, make sure that the gel completely soaked in electrophoresis buffer, vertically pull out the comb.
血清蛋白琼脂糖凝胶电泳(agarose gel electrophoresis)
血清蛋白琼脂糖凝胶电泳(agarose gel electrophoresis)【原理】琼脂糖（agarose）是经过挑选，以质地较纯的琼脂（agar）作为原料而制成的。
琼脂在化学上是由琼脂糖和琼脂胶组成的复合物。
琼脂胶是一含有硫酸根和羟基的多糖，它具有离子交换性质，这种性质会给电泳及凝胶过滤以不良的影响。
琼脂糖是直链多糖，它由D－半乳糖和3,6－脱水－L－半乳糖的残基交替排列组成。
琼脂糖主要通过氢键而形成凝胶。
电泳时因凝胶含水量大（98－99%），近似自由电泳，因为固体支持物的影响少，故电泳速度快，区带整齐。
而且由于琼脂糖不含带电荷的基团，电渗影响很少，是一种较好的电泳材料，分离效果较好。
血清中脂类物质与血清载脂蛋白结合成水溶性的脂蛋白（lipoprotein）形式存在。
各种脂蛋白中所含载脂蛋白的种类及数量不同，各种脂蛋白颗粒大小也相差很大，因此，以琼脂糖凝胶为支持物，在电场中可使各种脂蛋白颗粒分离开来。
琼脂糖凝胶电泳分离血清蛋白方法简单。
将血清脂蛋白用脂类染料苏丹黑（或油红等）进行预染。
再将预染过的血清加样于琼脂糖凝胶板加样槽中，通电后可以看到脂蛋白向正极移动，并分离出几个区带。
正常人血清脂蛋白可出现三条区带，从阴极到阳极依次为β－脂蛋白（最深），前β－脂蛋白（最浅）及α－脂蛋白（比前β－脂蛋白略深些），在原点处应无乳糜微粒。
有时前β－脂蛋白也显示不出来。
琼脂糖主要通过氢键而形成凝胶。
电泳时因凝胶含水量大（98－99%），近似自由电泳，因为固体支持物的影响少，故电泳速度快，区带整齐。
而且由于琼脂糖不含带电荷的基团，电渗影响很少，是一种较好的电泳材料，分离效果较好。
血清中脂类物质与血清载脂蛋白结合成水溶性的脂蛋白（lipoprotein）形式存在。
各种脂蛋白中所含载脂蛋白的种类及数量不同，各种脂蛋白颗粒大小也相差很大，因此，以琼脂糖凝胶为支持物，在电场中可使各种脂蛋白颗粒分离开来。
琼脂糖凝胶电泳分离血清蛋白方法简单。
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且结合后，能够极大增强荧光信号。
（四） 凝胶中DNA的成像
可以用透射或入射紫外光对EB染色的凝胶成
像，图像可以直接输出到计算机观察。
（五） 凝胶中DNA的回收
现一般采用试剂盒回收：存在的 Nhomakorabea要问题：
1 2 不能有效的回收大片段DNA 不能有效回收少量DNA
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二、 聚丙烯酰胺凝胶电泳
polyacrylamide gel electrophoresis
2 非变性聚丙烯酰胺凝胶
用于双链DNA片段的分离和纯化。 注：迁移率受其碱基组成和序列的影响。
用途：制备高纯度的DNA片段。
（三）DNA在聚丙烯胺凝胶中的有效分离范围
丙烯酰胺浓度 有效分离范围 （%） （bp） 二甲苯氰FF 溴酚蓝
标 准 (kb)
1~50 0.7~25 0.5~15 0.25~12 0.15~6 0.08~4
高强度 (kb)
低熔点 (kb)
低黏度低 溶点(kb)
0.8~10 0.4~8 0.3~7 0.2~4 0.1~3
0.8~10 0.4~8 0.3~7 0.2~4 0.1~3 0.05~1 0.5~1 0.1~0.5 0.01~0.1
4℃
IV
4℃
（三）琼脂糖凝胶中DNA的检测
通过染色, 紫外灯下检测。 主要有溴化乙锭（ethidium bromide, EB）染色法和SYBR Gold染色法。
1 凝胶的EB染色
使用EB染色注意事项
（1）EB被认为是一种强致癌物质
（2）EB可用来检测单链或双链核酸
（3）EB使用时的配制、贮存及使用
PAGE
（一） 聚丙烯酰胺凝胶的本质
在TEMED (四甲基乙二胺) 催化过硫酸铵还原 产生的自由基的存在下，丙烯酰胺单体的乙烯 基聚合形成聚丙烯酰胺的线状长链。
在双功能交联剂如N，N`-亚甲双丙烯酰胺的 参与下的共聚合反应中，聚丙烯胺的交联形成 三维带状网格结构。 网格孔径的平均直径决定于丙烯酰胺和双功 能交联剂的浓度。
EB常用水配制成10 mg/ml的贮存液，
于室温保存在棕色瓶或用铝箔包裹的瓶 中，使用终浓度为0.5 μg/ml 。
（4）当要知道DNA片段准确大小时，
凝胶应在无EB情况下电泳，电泳 结束后再用EB染色。
2 凝胶SYBR Gold的染色
SYBR Gold是一种新型极敏感染料的商
品名称。其与DNA结合的亲和力高，并
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一、 琼脂糖凝胶电泳
Agarose gel electrophoresis
（一）凝胶的制备及电泳
（二）DNA的迁移速率决定因素
1
DNA分子的大小
双链DNA分子迁移的速率与其碱基 对数的常用对数近似成反比； 2 琼脂糖浓度
浓度越低，相同核酸分子迁移越快；
3
DNA的构象
一般同一分子：超螺旋环状＞线状 ＞切口环状。 4 凝胶和电泳缓冲液中的溴化乙锭
CH2=CH-C(O)-NH2
(丙烯酰胺）
CH2=CH-CO-NH-CH2-NH-CO-CH=CH2 （N，N`-亚甲双丙烯酰胺）
（二） 聚丙烯酰胺凝胶电泳种类
1 变性聚丙烯酰胺凝胶
用于单链DNA片段的分离或纯化。
变性的DNA在这些凝胶中的迁移率几乎与其 碱基组成及序列完全无关。 用途：放射性DNA探针的分离、DNA测序反应等。
溴化乙锭（EB）插入双链DNA造成其 负电荷减少、刚性和长度增加。
5
所用的电压
低电压时DNA片段迁移率与所用 的电压成正比。
6
琼脂糖种类
常见的有两种：标准琼脂糖和低 熔点琼脂糖；
不同类型琼脂糖的性质
琼脂糖类型 凝结温度/℃ 35~38 40~42 熔化温度/ ℃ 90~95 85~90
标准琼脂糖 高强度琼脂糖
34~43 修饰的低熔点/ 25~35 凝点琼脂糖 35 8~15 超低熔点 25~30 低黏性低熔点琼 38 脂糖 30
85~95 63~65 65 40~45 70 85 75
不同厂家 生产的不 同商品其 凝结温度 和熔化温 度有一定 差异
不同类型琼脂糖分离DNA片段的范围
浓度 （%）
0.3 0.5 0.8 1.0 1.2 1.5 2.0 3.0 4.0 6.0
7
电泳缓冲液
常用的有TAE、TPE及TBE
TAE、TPE及TBE电泳缓冲液比较
都是常用电泳缓冲液。三者相比： 1）TAE的缓冲容量最低，如长时间电泳会被消耗，此时凝 胶的阳极一侧将发生酸性化； 2）TBE和TPE比TAE花费稍贵，但有高得多的缓冲容量；
3）双链线状DNA片段在TAE中比在TBE或TPE中迁移快10%；
第四节 核酸的凝胶电泳 Nucleic Acid Gel Electrophoresis
Content of Table
前 言 一、 琼脂糖凝胶电泳 二、 聚丙烯酰胺凝胶电泳 三、 脉冲场凝胶电泳
前 言
核酸凝胶电泳是分子克隆核心技术之一，
用于分离、鉴定和纯化DNA或RNA片段；
优点：（1）便于分离;（2）便于检测; （3）便于回收：
6×凝胶载样缓冲液
类型 I 6 ×缓冲液 0.25%溴酚蓝 0.25%二甲苯氰FF 4℃ 贮存温度
40% (m/V)蔗糖水溶液
0.25%溴酚蓝 II 0.25%二甲苯氰FF 15% Ficoll(Type400)水溶液 0.25%溴酚蓝 室温
III
0.25%二甲苯氰FF
30%甘油水溶液 0.25%溴酚蓝 40% (m/V)蔗糖水溶液
4）对于高分子质量的DNA，TAE的分辨率略高于TBE或TPE， 对于低分子质量的DNA，TAE要差些。超螺旋DNA在TAE 中的电泳分辨率要好于TBE。
（二） 凝胶载样缓冲液
载样缓冲液：临上样到凝胶加样孔之前与待 电泳的样品相混合的一种缓冲液。 载样缓冲液有三个作用：
1）增加样品密度保证DNA沉入加样孔内； 2）使样品带有颜色便于简化上样过程； 3）其中的染料在电场中以可以预测的泳动速 率向阳极迁移。
核酸凝胶电泳的基本原理
1）核酸分子之糖-磷酸骨架中的磷酸基团，呈负离子化状 态；核酸分子在一定的电场强度的电场中，它们会向正 电极方向迁移；
2）由于在电泳中往往使用无反应活性的稳定的支持介质， 电泳迁移率（或迁移速度）与分子的摩擦系数成反比。 而摩擦系数是分子大小、介质粘度等的函数；
因此，可在同一凝胶中、一定电肠强度下、可在凝胶 上分离出不同分子量大小或相同分子量但构型有差异的 核酸分子。