Name: polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies
Uploaded: 2012-05-22T12:00:00
Duration: 9 min
Description: Watch this Scientific Journal Video about Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies at JoVE.com

特别感谢在加州大学洛杉矶分校的克里斯Reddi设立试剂和浇注凝胶和艾琳·桑德斯在加州大学洛杉矶分校的灵感，指导和支持，并校对稿件。我还想感谢吉安卡洛Costaguta和格雷戈里·S·佩恩提供酵母基因组DNA和引物放大GAL3的基因。我还想感谢Bhairav​​沙阿拍照的实验室设备和试剂用于使数字2 - 4。霍华德休斯医学研究所（HHMI的批准号：52006944），为这个项目提供了资金。

<ol>
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我什么都没有透露。

PCR技术已成为不可或缺的工具，在生物科学宝库。 PCR技术已经改变了科学课程，让生物学家产生了基因组的力量，并具有新功能的混合基因，使具体和准确的临床试验，获得进入基因组和多样性，以及简单的克隆基因作进一步的生化分析的见解。 PCR的应用是有限的，只有挥动其权力的科学家的想象力。有许多书籍和文章描述的PCR新的专门用途，并有更多的将通过开发下一代生物科学。然而，无论预期的方法，基本框架已经保持不变。聚合酶链反应，在其所有的宏伟，是在体外生成一个特定的DNA片段的大量的应用。 设计PCR实验要求的思想和耐心。在图3所示的结果，体现的主要通道之一allenges设计时的PCR优化策略。也就是说，PCR扩增作为一个参数被改变，它可能会影响另一个。作为一个例子，如果初始PCR分最佳退火温度（58℃）进行了一个最佳的Mg 2 +浓度为2.0毫米，那么其结果将产生在图3c看到涂抹。试图解决涂片可能涉及设立含有2.0毫米MgCl 2的反应PCR条件和退火温度调整到61°C。然而，在图3a可见，这不会产生任何产品。因此，它是最好的试剂，滴定，而不是加入一个集中到一个单一的反应，故障排除时，杂散结果。此外，最常见的是调整优化PCR实验的要求是改变Mg 2 +浓度和纠正的退火温度。但是，如果这些变化不减少或废除异常的结果，additi滴定VES和/或改变循环条件如表2-6中所述的协议可能会缓解这一问题。如果一切都失败了，重新设计的引物和尝试，再试一次。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 的Taq聚合酶</td><td>西格玛</td><td> D4545-25UN </td><td></td></tr><tr><td> dNTPs浓度</td><td> Qiagen公司</td><td> 201225 </td><td></td></tr><tr><td> 0.2毫升薄壁管PCR管</td><td>生物拉德</td><td> 223-9469 </td><td></td></tr><tr><td> 0.2毫升薄壁管帽</td><td>生物拉德</td><td> 223-9472 </td><td></td></tr><tr><td> TE缓冲液： </td><td></td><td></td><td></td></tr><tr><td> EDTA二钠盐二水</td><td>西格玛</td><td> E5134 </td><td></td></tr><tr><td> trizma-盐酸</td><td>西格玛</td><td>的T-3253 </td><td></td></tr><tr><td>自定义噬菌体底漆</td><td> Invitrogen公司</td><td colspan="2"> 25441F_RL6_Contig2_68k TACTGCAACGCGATGTTGCG </td></tr><tr><td>自定义噬菌体底漆</td><td> Invitrogen公司</td><td colspan="2"> 26007R_RL6_ Contig2_68k TCACCATCAATCGTGGCGGT </td></tr><tr><td>自定义酵母底漆</td><td>集成DNA技术</td><td colspan="2"> SACI-GAL3（-256）ACCTGGAGCTCCTATTT TAACATGTGGATTTCTTGAAAGAATGAAATCG </td></tr><tr><td>自定义酵母底漆</td><td>集成DNA技术</td><td colspan="2"> GAL3（1848年），SACI CGGATGGAGCTCGCGT GAAGGTAATTTTTTTTTATGTCTCCG </td></tr><tr><td>热循环仪</td><td>生物拉德</td><td> 170-9703 </td><td>我的基因扩增仪</td></tr><tr><td>琼脂糖</td><td> ISC的BioExpress </td><td> E-3120-500 </td><td></td></tr><tr><td>凝胶电泳</td><td> hoeffer科学仪器</td><td>型号HE33 </td><td></td></tr><tr><td> 1 kb的DNA梯状</td><td> New England Biolabs公司</td><td> N3232S </td><td></td></tr><tr><td>生物RAD电源包</td><td>生物拉德</td><td> 164-5050 </td><td> PowerPac的基本</td></tr><tr><td>凝胶文件系统</td><td> fotodyne法团</td><td colspan="2"> FOTO /调查分析师FX工作站</td></tr></tbody></table>

polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies

在生物科学，有技术进步的纪律弹射到发现的黄金年龄。例如，微生物学领域的转化与安东·范·列文虎克的显微镜，它允许科学家们想象的首次原核生物的到来。聚合酶链反应（PCR）的发展是一个创新，改变其影响跨越无数的分支学科，在生物分子科学当然。理论的过程概述了由Keppe和同事在1971年，然而，它是14年，直到完整的PCR过程描述和实验由Kary穆利斯应用，而在鲸鱼座公司于1985年。自动化和完善这一技术进步与引进菌嗜水生 ，因此得名Taq DNA聚合酶的热稳定的DNA聚合酶。 PCR是1鲍威RFUL扩增技术，可以产生一个特定的DNA（即扩增）段供应充足，只有少量的起始原料（即模板DNA或靶序列）。虽然简单，一般无故障，也有复杂的化学反应产生的杂散结果的陷阱。 PCR扩增失败时，它可以导致许多大小不等的非特异性的DNA琼脂糖凝胶带梯子或涂片中出现的产品。有时，没有产品形成所有。另一个潜在的问题发生突变时无意中引入的扩增，在PCR产物的异构人口。 PCR扩增失败可以成为令人沮丧的，除非耐心和细心的故障排除理清和解决的问题（S）。该协议概述了PCR技术的基本原则，提供了一种方法，这将导致大多数靶序列的扩增，并提出了优化反应的战略。按照这样的PCR指南，STudents应该能够： <ul><li>设立常规PCR实验反应和热循环条件</li><li>了解各种反应元件的功能及其在PCR实验的整体效果</li><li>设计和优化为任何DNA模板的PCR实验</li><li>失败的PCR实验疑难解答</li></ul>

Watch this Scientific Journal Video about Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies at JoVE.com

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

PCR技术已经成为了许多分子生物学实验室常用技术。这里提供几个常规PCR协议是一个快速指导。因为每一个反应是一个独特的试验，以产生不同产品所需的最佳条件。了解反应中的变量，将大大提高故障排除效率，从而增加了机会，以获得所需的结果。

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Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

复用荧光免疫检测方法和故障排除

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

亚克隆加插入（SPI） - 一种新型重组工程方法基因打靶载体的快速构建

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

全自动毛细 Electrophoretic-based 免疫分析方法的程序和关键优化策略

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

中子大分子晶体学晶体生长优化

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX ...