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。

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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>

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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。

聚合酶链反应：基本协议加上故障排除和优化策略

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

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Biology

403.5K 次观看.  University of California, Los Angeles. 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。

视频: Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

核苷三磷酸 - 从合成到生化特征

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

科研

化学

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the traditional method of choice is 3&#39; rapid amplification of cDNA ends (3&#39; RACE). Protocols for 3&#39; RACE require the careful design and selection of nested primers within the 3&#39; untranslated region (3&#39; UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3&#39; UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3&#39; UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3&#39; RACE. Expanded 3&#39; RACE can detect unusual 3&#39; UTRs, including gene fusions within the 3&#39; UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

The two different 3&#39; rapid amplification of cDNA ends (3&#39; RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3&#39; UTR of a transcript using RNA obtained from different cancer cell lines.

3 ' 快速扩增 CDNA 末端, 以图记录癌症

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

遗传学

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

使用含有微型基因的Alu元素来分析圆形RNA

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

生物化学

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

引物延伸捕获：从严重退化的DNA来源有针对性的顺序检索

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

生物学

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

使用无细胞系统对用于蛋白质原型的最小线性模板进行扩增的快速酶促方法

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...

生物工程

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

使用无细胞系统快速表征遗传部分

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A &#946;-estradiol-inducible AID system was created, with expression of TIR1 under the control of a &#946;-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with &#946;-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate &#946;-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the &#946;-est AID system.

调整降解以实现特定和高效的蛋白质消耗

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...

免疫与感染

PCR: The Polymerase Chain Reaction

The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling &#8211; cyles of temperature changes at fixed time intervals. Using a thermostable DNA polymerase, PCR can create numerous copies of DNA from DNA building blocks called dNTPs. There are&#160;three steps in PCR: denaturation, annealing, and elongation. Denaturation is the first step in the cycle and causes the DNA to melt by disrupting hydrogen bonds between the bases resulting in single-stranded DNA. Annealing lowers the temperature enough to allow the binding of oligonucleotide primers to the DNA template. During the elongation step DNA polymerase will synthesize new double-stranded DNA.
This video provides an introduction to the PCR procedure. The basic principles of PCR are described as well as a step-by-step procedure for setting up a generalized PCR reaction. The video shows the necessary components for a PCR reaction, includes instruction for primer design, and provides helpful hints for ensuring successful PCR reactions.

PCR: Principle, Instrumentation, and Applications

PCR: 聚合酶链式反应

The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling &#8211; cyles of temperature changes at fixed time ...

教育

JoVE Science Education

基础生物学

细胞分子生物学中的基本方法

Detecting Environmental Microorganisms with the Polymerase Chain Reaction and Gel Electrophoresis

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University 
Demonstrating Author: Bradley Schmitz
Polymerase chain reaction (PCR) is a technique used to detect microorganisms that are present in soil, water, and atmospheric environments. By amplifying specific sections of DNA, PCR can facilitate the detection and identification of target microorganisms down to the species, strain, and serovar/pathovar level. The technique can also be utilized to characterize entire communities of microorganisms in samples.
The culturing of microorganisms in the laboratory using specialized growth media is a long-established technique and remains in use for the detection of microorganisms in environmental samples. Many microbes in the natural environment, while alive, maintain low levels of metabolic activity and/or doubling times and are thus referred to as viable but non-culturable (VBNC) organisms. The use of culture-based techniques alone cannot detect these microbes and, therefore, does not provide a thorough assessment of microbial populations in samples. The use of PCR allows for the detection of culturable microbes, VBNC organisms, and those that are no longer alive or active, as the amplification of genetic sequences does not generally require the pre-enrichment of microorganisms present in environmental samples. However, PCR cannot differentiate the aforementioned states of viability and activity. When combined with one or more culture-based techniques, the viability of certain subsets of microorganisms may still be determined.

Detecting Environmental Microorganisms Using PCR

聚合酶链反应与凝胶电泳检测环境微生物

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz
Polymerase chain reaction (PCR) is ...

环境科学

环境微生物学

Gradient Polymerase Chain Reaction to Determine the Optimum Annealing Temperature

Source: Tsujimoto, H. et al., <a href="https://app.jove.com/v/63008">Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti.</a> J. Vis. Exp. (2021).
This video demonstrates the gradient polymerase chain reaction for optimization of the annealing temperature of primers, which sets gradient annealing temperature along wells. The effect of different annealing temperatures is determined by the yield of amplified products that helps to optimize the reaction condition.

梯度聚合酶链式反应，用于确定最佳退火温度

Source: Tsujimoto, H. et al., Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti. J. Vis. ...