Name: streamlined purification of plasmid dna from prokaryotic cultures
Uploaded: 2011-01-05T17:00:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

1。裂解液间隙<ol><li>成长大肠杆菌改造所需的质粒DNA在深孔板（1毫升文化/）卢里亚肉汤与适当的抗生素一夜之间在37℃用颤抖的彗星的大肠杆菌培养。 </li><li>佩莱大肠杆菌的培养板在5000 10分钟的XG，然后倒出上清液。 </li><li>每100μL，再悬浮缓冲液（50毫米的Tris - HCl，pH值8.0，10 mM的EDTA，100微克/毫升RNA酶A）重悬沉淀。 </li><li>加入100μL/孔裂解液（200毫米氢氧化钠，1％SDS）和摇板振动筛2分钟。 </li><li>加入100μL/孔和缓冲液（3.0 M醋酸钾，pH值5.5），振摇2分钟。 </li><li>转移细胞裂解液，裂解液澄清板。 </li><li> DNA结合的板350μL收集板和成真空歧管底部的地方上。组装真空歧管（见图1）。 </li><li>裂解液放置澄清真空装置上板。 </li><li>应用在10英寸汞柱（0.34巴）真空，滤液收集到的DNA结合板。不要使用大于12英寸汞柱真空。 （0.41 bar）的。 </li><li>拆开真空歧管。将2毫升废物收集板装置的底部。 </li></ol> 2。 DNA结合<ol><li>移动的DNA结合板多方面的顶部（见图2）。 </li><li>加入300μL/孔约束力的缓冲区（6 M盐酸胍）和吸管向上和向下混合。 </li><li>应用真空〜5英寸汞柱（0.17巴）缓慢真空和丢弃滤液。 DNA是现在势必膜。 </li><li>与400μL洗涤缓冲液（80％乙醇）/清洗。 </li><li>应用真空，然后丢弃滤液。如果使用2毫升收集板或过滤直接浪费丢弃不必要的。 </li><li>重复洗一次。 </li><li>吸干底部过滤板吸水毛巾擦干。 </li><li>应用真空一次5-10分钟，以确保去除残留的异丙醇。 </li><li>吸干底部，以确保去除乙醇滴。 </li></ol> 3。 DNA的洗脱<ol><li>添加70μL/孔洗脱缓冲液（10毫米的Tris - HCl，pH值8.0，1毫米EDTA）。如果需要较高的DNA浓度，体积可减少到50μL/孔，但平均总收益率将降低。 </li><li>一分钟，在室温下孵育板。 </li><li>可无论是真空或离心洗脱纯化的DNA。 </li><li>真空法：将清洁的收集板（脱氧核糖核酸酶，核糖核酸酶自由）进入真空系统。放在真空歧管上的过滤板，适用于15英寸汞柱（0.5巴）1分钟真空，直到所有的洗脱缓冲液通过DNA结合板通过。收集纯化后的DNA（见图3）。 </li><li>离心法：将清洁收集板的顶部和1000 XG 5分钟离心净化过滤板。 </li></ol> 4。定量和定性分析<ol><li>测量外径260/280，计算纯化的DNA的浓度。 </li><li>进行了纯化的质粒DNA琼脂糖凝胶电泳分析。 </li></ol> 5。代表性的成果 大肠杆菌裂解液澄清使用传统的离心和帕尔AcroPrep提前裂解液澄清过滤板显示的碎片明显减少，从起始原料。 （图4）。 到TE缓冲液中加入三个浓度的质粒DNA，并通过AcroPrep提前裂解液澄清过滤板通过。质粒DNA浓度的比较，前，后过滤，显示在所有三个浓度〜100％的恢复（图5）。 为了证明澄清大肠杆菌的DNA恢复添加到原油裂解大肠杆菌裂解，质粒DNA，然后通过过滤澄清。相同的质粒DNA加入到细胞裂解液以下过滤。电泳是从每个样本（图6）等分。一个单一的，从每个样品类似强度的明确乐队演示一点在澄清过滤步骤如有DNA损失。 文化的E.从 E 大肠杆菌含有PCAT质粒DNA纯化大肠杆菌裂解液使用颇尔公司的350μL和1毫升的DNA结合过滤板，以及两个其他品牌的350μL的DNA结合板。所有纯化制造商建议的协议规定执行。颇尔公司的1毫升AcroPrep进展DNA纯化过滤板（图7和表2）从最高的DNA浓度和总收率。 虽然竞争对手W也显示了DNA浓度高（155毫微克/μL），总DNA的产量是最低的（4.7微克/孔），由于回收量低，〜30微升。竞争者中号了108纳克/μL总产的5.6微克/以及DNA的最低浓度。 洋菜OSE凝胶纯化从三种不同的DNA结合板PCAT DNA分析，如图8所示。每次采集的样本量调整至65μL，以正确的回收量的差异，以电泳前。所有样品超螺旋质粒DNA，但每个滤板的DNA收益率各不相同。 图9显示了所有准备的DNA超螺旋的形式，也有类似的的浓度。 <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="图1" /> 图1。大会歧管的真空裂解液澄清 。 <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="图2" /> 图2。大会的DNA结合真空歧管 。 <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="图3" /> 图3。大会真空歧管真空洗脱。 <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="图4" /> 图4。过滤简化了样品澄清。一式三份样品300μL真空过滤（350微升AcroPrep提前裂解液澄清过滤板）或传统的离心澄清原油裂解。外径320测量之前和之后的澄清。 <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="图5" /> 图5。 PDNA通过后，通过AcroPrep提前裂解液澄清过滤板的全面恢复。 300μLTE缓冲液中添加与PCAT在25，50和100微克/毫升。 DNA浓度和恢复计算外径260过滤前后，N = 2。 <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="图6" /> 图6。 PCAT的DNA后的恢复澄清 。裂解液之前或之后澄清飙升的pUC19相等，则稀释1至10日在TE缓冲液。装2微升/ 1.2％琼脂糖凝胶上的车道。 <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="图7" /> 图7。质粒DNA，每口井产量与PALL Acroprep进展DNA结合比竞争对手板过滤板 。PCAT质粒DNA产量（ 外径 260）使用1毫升（帕尔和竞争对手中号）或1.5毫升（竞争对手宽）过夜培养的指示的DNA纯化板DH5α中。净化用板制造商的推荐协议。误差线表示标准错误（N&gt; 6）。 <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="图8" /> 图8。琼脂糖凝胶电泳分析PCAT的DNA纯化表示DNA结合板 。 1.2％，纯化的质粒DNA琼脂糖凝胶电泳。由三个独立的净化池的样品，洗脱液净化后，稀释1:10调整至65μL。装2％μL巷。 <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="图9" /> 图9。纯DNA相似的离心或真空方法最终洗脱液收集纯化的质粒DNA琼脂糖凝胶电泳时的质量和恢复。其次PDNA净化AcroPrep预先过滤板制造商的建议协议。洗脱在1000 XG离心5分钟或2分钟与15英寸汞柱（0.5巴）的真空。

此视频文章的作者是谁生产仪器和试剂的视频和文字的特色，颇尔生命科学的员工。

我们描述了一个浅显的，精简AcroPrep提前滤板的协议，给予几个优点。在手工，半自动化或全自动化AcroPrep提前格式的处理时，这种方法提供了最大的质粒DNA产量。对裂解间隙板的集成预过滤，提供样品，即使是那些含有较高水平的微粒过滤一致。更快，更均匀，整个板块的滤过率，以最小的滞留量板的结果以及几何;和出口尖几何提供了直接到接收板样品流，无交叉污染的关注。更重要的是，高的DNA结合能力AcroPrep预先过滤板具有约束力板膜是优化高质量的质粒DNA的最大恢复。

<table><tbody><tr><td>AcroPrep Advance Filter plate</td><td>Pall Corporation</td><td>PN 8175 (1 mL) or PN 8075 (350 &amp;#181;l)</td><td>3 &amp;#x03BC;m glass fiber 0.2 &amp;#x03BC;m Supor&amp;#174; membrane for clarification</td></tr><tr><td>AcroPrep Advance Filter Plate</td><td>Pall Corporation</td><td>PN 8132</td><td>for DNA Binding</td></tr><tr><td>Vacuum manifold</td><td>Pall Corporation</td><td>PN 5017</td><td></td></tr><tr><td>Vacuum pressure pump</td><td>Pall Corporation</td><td>PN 13157</td><td></td></tr></tbody></table>

streamlined purification of plasmid dna from prokaryotic cultures

我们描述AcroPrep提前96个质粒的准备滤板的完整的过程，从原核文化，并以高纯度的DNA结尾。基于多井过滤细菌裂解液检查和DNA纯化，这种方法创建一个质粒编制的精简过程。含有硅为基础的媒体的过滤板可以很容易地处理，真空过滤或离心产生可观数量的质粒DNA。定量分析确定纯化的质粒DNA是高品质的一贯平均OD 260/280比值1.97 。总体而言，质粒产量提供更纯净的DNA测序和克隆，下游应用，如。这种简化方法采用AcroPrep提前滤板允许手动，半自动或全自动化处理。

Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

此协议是一个成本效益的替代方案，为高效的并行澄清和质粒DNA纯化<em&gt; E。大肠杆菌</em&gt;文化。 AcroPrep发展的过程开始于一个优化的裂解液澄清过滤净化结合能力高的DNA结合滤板的车牌。

从原核文化精简纯化的质粒DNA

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

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Research

JoVE Journal

Biology

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视频: Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

科研

生物化学

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

生物学

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

免疫与感染

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

在培养的哺乳动物细胞大规模并行报告基因分析

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Plasmid Stability Analysis with Open-Source Droplet Microfluidics

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy. This approach allows for the parallel analysis of numerous droplets and microcolonies, providing greater statistical power compared to traditional plate counting and enabling the integration of the assay into other droplet microfluidic workflows. By using plasmids expressing fluorescent proteins alongside a non-specific fluorescent DNA stain, single colonies can be identified and differentiated based on plasmid loss or fluorescent marker expression. Notably, this advanced workflow, implemented with open-source hardware, offers precise flow control and temperature management of both the sample and the microfluidic chip. These features enhance the workflow&#39;s ease of use, robustness, and accessibility. While the study focuses on Escherichia coli as the experimental model, the method&#39;s true potential lies in its versatility. It can be adapted for various studies requiring fluorescence signal quantification from plasmids or stains, as well as for other applications. The adoption of open-source hardware broadens the potential for conducting high-throughput bioanalyses using accessible technology in diverse research settings.

An accessible, open-source microfluidic workflow is presented for the parallelized analysis of plasmid retention in bacteria. By employing fluorescence microscopy to quantify plasmid presence in single-cell microcolonies within gel microdroplets, this method provides a precise, accessible, and scalable alternative to traditional plate counting.

使用开源液滴微流控进行质粒稳定性分析

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating ...

生物工程

Use of In Vivo Assembly for High-efficiency Plasmid Construction

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA fragments to assemble plasmids. This method functions by transforming DNA fragments with regions of 15-50 bp of homology into commonly used laboratory Escherichia coli strains and the bacteria use the RecA-independent repair pathway to assemble the DNA fragments into a plasmid. This method is more rapid and cost-effective than many molecular cloning methods that rely on in vitro assembly of plasmids prior to transformation into E. coli strains. This is because in vitro methods require the purchase of specialized enzymes and the performance of sequential enzymatic reactions that require incubations. However, unlike in vitro methods, IVA has not been experimentally shown to assemble linear plasmids. Here we share the IVA protocol used by our laboratory to rapidly assemble plasmids and subclone DNA fragments between plasmids with different origins of replication and antibiotic resistance markers.

In vivo assembly is a ligation-independent cloning method that relies on intrinsic DNA repair enzymes in bacteria to assemble DNA fragments by homologous recombination. This protocol is both time and cost-effective, as few reagents are required, and cloning efficiency can be as high as 99 %.

体内 组装用于 高效质粒构建

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

质粒来源的DNA链置换盖茨实施化学反应网络

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

从真核细胞染色体外的环状DNA基因组范围内的净化

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

CAPRRESI:通过质粒恢复和限制酶位点插入的嵌合体装配

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...

从原核文化精简纯化的质粒DNA

副本

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此视频中的章节

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

高通量的亲和标签的重组蛋白的纯化

在培养的哺乳动物细胞大规模并行报告基因分析

使用开源液滴微流控进行质粒稳定性分析

体内组装用于高效质粒构建

质粒来源的DNA链置换盖茨实施化学反应网络

从真核细胞染色体外的环状DNA基因组范围内的净化

CAPRRESI:通过质粒恢复和限制酶位点插入的嵌合体装配

从原核文化精简纯化的质粒DNA

副本

摘要

探索更多视频

此视频中的章节

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

高通量的亲和标签的重组蛋白的纯化

在培养的哺乳动物细胞大规模并行报告基因分析

使用开源液滴微流控进行质粒稳定性分析

体内 组装用于 高效质粒构建

质粒来源的DNA链置换盖茨实施化学反应网络

从真核细胞染色体外的环状DNA基因组范围内的净化

CAPRRESI:通过质粒恢复和限制酶位点插入的嵌合体装配

体内组装用于高效质粒构建