实验十 PCR扩增DNA

实验十 PCR扩增DNA 实验三 PCR扩增DNA 【实验目的】 1( 掌握PCR扩增DNA的技术及原理 2( 学习PCR扩增仪的使用 【实验原理】 聚合酶链反应(polymerase chain reaction，PCR)是一种体外DNA扩增技术，1985年由Mullis等人创立。该技术能在几小时的实验操作中，将人为选定的一段DNA扩增几百万倍，具有灵敏度高、特异性强、操作简便和应用广泛等优点，目前已成为分子生物学及基因工程中极为有用的研究手段，另外在医学研究和医疗诊断中亦体现出极大的应用价值。 PCR的工作原理类似于DNA的体内复制过程，是将待扩增的DNA片断和与其两侧互补的寡核苷酸链引物经变性、退火及延伸三步反应的多次循环，使特定的DNA片断在数量上呈指数增加。PCR扩增首先需要一对引物，根据待扩增区域两侧的已知序列合成两个与 模板 个人简介word模板免费下载关于员工迟到处罚通告模板康奈尔office模板下载康奈尔 笔记本 模板 下载软件方案模板免费下载 DNA互补的寡核苷酸作为引物，引物序列将决定扩增片段的特异性和片段长度。反应体系由模板DNA、一对引物、dNTP、耐高温的DNA聚合酶、酶反应缓冲体系及必须的离子等所组成。PCR反应循环的第一步为加热变性，使双链模板DNA变性为单链;第二步为复性，每个引物将与互补的DNA序列杂交;第三步为延伸，在耐高温的DNA聚合酶作用下，以变性的单链DNA为模板，从引物3ˊ端开始按5ˊ?3ˊ方向合成DNA链。这样经过一个周期的变性——复性——延伸等三步反应就可以产生倍增的DNA，假设PCR的效率 n为100%,反复n周期后，理论上就能扩增2倍。PCR反应一般30-40次循环，DNA片段可放大数百万倍。 常规PCR反应用于已知DNA序列的扩增，变性温度为95?，复性温度为37?,55?，延伸合成温度为72?，DNA聚合酶为Taq酶(可耐受95?左右的高温而不失活)，反应循环数为30。 【实验材料】 1. 实验器材 PCR扩增仪，台式高速离心机，移液器，经高压灭菌后的Eppendorf管，电泳仪。 2.实验试剂 (1)模板DNA(0.1µg/µl):用牙签挑取单菌落悬浮到50µl的裂解液中(裂解液1%TritonX-100，20mmol/l Tris-HCl PH 8.2, 2mmol/l EDTA), 95?温浴10分钟, 10000rpm离心5分钟,取2µl上清液用于总体积50µl的PCR反应。 (2)TaqDNA聚合酶(5U/µl)，10×扩增缓冲液，dNTP(1mmol/L):购买 (3)引物(100pmol/L):设计并合成与目的DNA两侧互补的引物内。 【实验操作】 1(准备PCR反应溶液 (1) 按以下次序，将下列成分在0.5ml灭菌Eppendorf管内混合: 10×扩增缓冲液 10µl 4×dNTPs(包括四种dNTP) 8µl 引物1 1µl 引物2 1µl 模板DNA 1µl (10ng) Taq DNA聚合酶 1µl (2.5U) 加水至终体积 100µl (2) 用手指轻弹Eppendorf管底部，使溶液混匀。在台式离心机中离心2秒以集中溶 液于管底。 (3)加石蜡油50µl封住溶液 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 面。 2(PCR扩增反应 将加好样品的Eppendorf管置于PCR扩增仪内，95?5分钟，使模板DNA完全变性。然后按95?变性1分钟，55?退火45秒，72?延伸1分钟，重复循环30次，循环结束后72?延伸8分钟。反应完毕，将样品取出置-4?待用。 【实验结果】 取出样品，进行琼脂糖凝胶电泳或聚丙烯酰胺电泳，溴化乙锭(EB)染色，观察DNA条带。具体操作方法见实验(6)。 【讨论】 1(PCR结果若出现非特异性的扩增条带，有必要进一步优化反应条件，包括改变退火温 2+度和时间，调整Mg浓度等。 2(PCR反应特异性强，引物浓度、TaqDNA聚合酶和dNTP的量不宜过多。 3(引物设计要合理。一般引物长度为18个~30个核苷酸;引物间的G + C含量应为40%,60%，而且避免引物内部产生二级结构;引物3ˊ端不应该互补，避免在PCR反应过程中产生引物二聚体;避免引物3ˊ端出现3个连续的G或C;理想的情况下，成对引物的G、C含量应相似以便以相近的退火温度与互补的模板链相结合，此外，引物5ˊ端序列对于后续操作也是十分有用的，例如，对PCR产物进行克隆时，可以考虑在引物5ˊ端引入限制性酶切位点。 【思考 题 快递公司问题件快递公司问题件货款处理关于圆的周长面积重点题型关于解方程组的题及答案关于南海问题 】 影响PCR的因素有哪些, Experiment 15. PCR Amplify DNA 【Purpose】 1、 Master the principle and the techniques of PCR Amplifying DNA 2、Learn how to use the PCR thermal cycler 】 【Principle Ploymerase chain reaction (PCR) is a technique of amplifying DNA in vitro, which was previously established in 1985 by Mullis et al. The technique, by which a fragment of DNA can be amplified many million-fold in a few of hours under experimental operation, has the advantages of high sensitivity，high specificity, ease of operation, extensive application and so on. Until now PCR has become an essential tool not only in Molecular Biology but also in genetic engineering. Furthermore，its applications are finding their way into many areas of medical research and medical diagnosis. The principle of PCR is similar to the natural process of DNA replication. The DNA fragment is amplified with two oligonucleotide primers，each complementary to one end of the DNA target sequence，in a three-step reaction of denaturing, annealing and polymerization. Many cycles can produce the definite DNA fragments increased in quantity by index. A pair of oligonucleotides will serve as PCR primers, which can be designed according to the known sequence information of template DNA .The primers are complementary to one end of the DNA target sequence so that they can decide the length and the location of the amplified DNA fragment. The reaction system comprises template DNA, a pair of primers, dNTPs, thermostable DNA polymerase, reaction buffer，magnesium and optional additives. Each cycle of PCR is as follows: Firstly , denaturation— Heat to denature the duplex DNA template at high temperature so that the target DNA is separated into two strands; Secondly, annealing— Decrease the temperature of the solution to make the primers and DNA template complement and anneal to partly form double chains; Finally, extension—With thermostable DNA polymerase , the single strand of the DNA template, which is obtained by denaturation, distends to be double strands by addition of ,nucleotides to primers 3ˊend. As a result, by reaction cycle of three steps: denaturation, primer annealing and polymerization ,the copy of the definite DNA fragment is doubled over and over nagain with each cycle. If PCR is 100% efficient, one target molecule would become 2 after n cycles. Usually by 30,40 cycles, DNA fragment is amplified by some million times. The basic PCR is applied to amplify those DNA fragments whose sequence has been known. The reaction cycle comprises a 95? step to denature the duplex DNA, an annealing step of around 37?,55? (the actual temperature depends on the primer lengths and sequences.) to allow the primers to bind and a 72? polymerization step. Taq DNA polymerase is usually used as thermostable DNA polymerase in PCR, which still survives the denaturation step of 95?. In addition, 30 cycles are used in the basic PCR. 【Materials】 1. Apparatus: PCR thermal cycler，Table high-speed centrifuger，Pipettor，Eppendorf tubes sterilized by high pressure，Electrophoresis apparatus. 2. Reagent: (1)Template DNA，0.1µg/µl:: Pick up single colony and suspend it in 50µl lysis solution( 1%TritonX-100，20mmol/LTris.Cl PH 8.2, 2mmol/L EDTA), put it in a warm bath of 95? for 10 minutes, then centrifuge at 10000 rpm for 5 minutes and remove the supernatant of 1µl to be used to PCR whose total reaction volume is 50µl. (2)Purchase Taq DNA polymerase(5U/µl)，10×amplification buffer，dNTPs solution containing all four dNTPs(1mmol/L) (3)Primers，100pmol/L:Design and synthesis primers each complementary to one end of the target DNA. 【Procedures】 1( Preparation of PCR reaction solution (1)In a sterile 0.5ml Eppendorf tube, mix in the following order: 10×amplification buffer 10µl solution of four dNTPs 4µl primer 1 1µl primer 2 1µl DNA template 2µl(10ng) Taq DNA polymerase 1µl(2.5U) Add water to terminal volume of 50µl (2)Pellet the bottom of the Eppendorf tube with finger gently in order to mix the solution well. Then the solution is collected by being put into the table high-speed centrifuger. (3)Overlay the reaction mixtures with 50µl of paraffin. 2(The process of PCR The Eppendorf tubes containing the reaction mixtures are put into the PCR thermal cycler, the reaction mixtures are kept at 95? for 5 minutes to denature DNA fully., Next in turns of denaturing at 95? for 1 minute ,annealing at 55? for 45 seconds and extending at 72? for 1 minute, repeat for 30 cycles. After these cycles are finished, the reaction mixtures are extended at 72? for a further 8 minutes. Finally，take out the sample tubes and store at -4?. 【Results】 Withdraw the samples and analyze them by electrophoresis through an agarose or polyacrylamide gel. Stain the gel with ethidium bromide to visualize the DNA. For the instruction in further details, please see 《Experiment 6 》. 【Discussion】 1( If PCR produces some nonspecific amplification, it may be necessary to optimize the reaction conditions. The parameters to be varied include the annealing temperature, the annealing 2+time and the Mgconcentration. 2( PCR has high specificity，so the concentration of the primes、Taq polymerase and dNTP should not be too high. 3( Design the primes rationally. PCR primers generally range in length from 18-30 bases. Primers should contain 40%,60% G，C and care should be taken to avoid sequences which would produce internal secondary structure. The 3ˊends of the primers should not be complementary to avoid the production of primer-dimers in the PCR reaction. Avoid three G or C nucleotides in a row near the 3ˊend of the primer. Ideally, both primers should have similar G， C content so that they anneal to their complementary sequences at similar temperatures. Additionally, the sequence of the primers can also include regions at the 5ˊend which can be useful for downstream applications. For example, restriction enzyme sites can be placed in the primer pair design if the desired PCR product is to be subsequently cloned. 【Advisement after experiment】 What is the effect of PCR? 莇衿腿莅薂袅膈蒇蒅螁膈膇蚁蚇膇艿蒃羅膆莂虿袁芅蒄蒂螇芄膄蚇蚃芃芆蒀羂节蒈 蚅羈节薁薈袄芁芀螄螀袇莂薇蚆袆蒅螂羄袆膄薅袀羅芇螀螆羄荿薃蚂羃薁莆肁羂芁蚁羇羁 莃蒄袃羀蒆蚀蝿羀膅蒃蚅罿芈蚈羄肈莀蒁衿肇蒂蚆螅肆节葿螁肅莄螅蚇肄蒇薇羆肄膆螃袂 肃芈薆螈膂莁螁蚄膁蒃薄羃膀膃莇衿腿莅薂袅膈蒇蒅螁膈膇蚁蚇膇艿蒃羅膆莂虿袁芅蒄蒂 螇芄膄蚇蚃芃芆蒀羂节蒈蚅羈节薁薈袄芁芀螄螀袇莂薇蚆袆蒅螂羄袆膄薅袀羅芇螀螆羄荿 薃蚂羃薁莆肁羂芁蚁羇羁莃蒄袃羀蒆蚀蝿羀膅蒃蚅罿芈蚈羄肈莀蒁衿肇蒂蚆螅肆节葿螁肅 莄螅蚇肄蒇薇羆肄膆螃袂肃芈薆螈膂莁螁蚄膁蒃薄羃膀膃莇衿腿莅薂袅膈蒇蒅螁膈膇蚁蚇 膇艿蒃羅膆莂虿袁芅蒄蒂螇芄膄蚇蚃芃芆蒀羂节蒈蚅羈节薁薈袄芁芀螄螀袇莂薇蚆袆蒅螂 羄袆膄薅袀羅芇螀螆羄荿薃蚂羃薁莆肁羂芁蚁羇羁莃蒄袃羀蒆蚀蝿羀膅蒃蚅罿芈蚈羄肈莀 蒁衿肇蒂蚆螅肆节葿螁肅莄螅蚇肄蒇薇羆肄膆螃袂肃芈薆螈膂莁螁蚄膁蒃薄羃膀膃莇衿腿 莅薂袅膈蒇蒅螁膈膇蚁蚇膇艿蒃羅膆莂虿袁芅蒄蒂螇芄膄蚇蚃芃芆蒀羂节