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. Apuramento lisado <ol><li> Crescer E. culturas coli transformada com plasmídeo de DNA desejado no bem profundo placas (1 mL da cultura / poço) em caldo de Luria com antibiótico adequado durante a noite a 37 ° C com agitação. </li><li> E. coli Pellet em placa de cultura em 5.000 xg por 10 minutos, em seguida, o sobrenadante decantar. </li><li> Ressuspender cada pellet em 100 mL de buffer ressuspensão (50 mM Tris-HCl, pH 8,0, 10 mM EDTA, 100 mcg / mL de RNase A). </li><li> Adicionar 100 mL Tampão de Lise / poço (200 mM NaOH, 1% SDS) e agitar com placa shaker por 2 minutos. </li><li> Adicionar 100 mL de buffer Neutralização / poço (3,0 M acetato de potássio, pH 5.5) e agitar por 2 minutos. </li><li> Transferência lisado celular para lisado placa esclarecimento. </li><li> Colocar a placa de ligação ao DNA em cima de 350 mL prato de coleta e coloque em baixo do colector de vácuo. Montar coletor de vácuo (ver Figura 1). </li><li> Coloque placa de esclarecimento lisado em cima do aparelho de vácuo. </li><li> Aplicar vácuo a 10 pol Hg (0,34 bar) e coletar DNA filtrado para placa de ligação. Não use vácuo superior a 12 pol Hg. (0,41 bar). </li><li> Desmonte coletor de pó. Coloque 2 mL placa de coleta de lixo no fundo do aparelho. </li></ol> 2. DNA Binding <ol><li> Mover a placa de DNA de ligação ao topo do colector (ver Figura 2). </li><li> Adicionar 300 mL de buffer / poço Binding (6 M de guanidina-HCl) e uma pipeta cima e para baixo para misturar. </li><li> Aplicar vácuo [~ 5 pol Hg (0,17 bar) para vácuo lento] e descartar filtrado. DNA é agora obrigado a membrana. </li><li> Lavar com 400 mL / poço de tampão de lavagem (80% de etanol). </li><li> Aplicar vácuo, em seguida, descartar o filtrado. Descarte desnecessário se usar duas placas coleção mL ou filtragem diretamente para o lixo. </li><li> Repita lave uma vez. </li><li> Blot fundo da placa de filtro de papel absorvente para secar. </li><li> Aplicar vácuo mais uma vez por 5-10 minutos para assegurar a remoção de resíduo de álcool. </li><li> Blot fundo para assegurar a remoção de gotículas de etanol. </li></ol> 3. Eluição DNA <ol><li> Adicionar 70 mL tampão de eluição / poço (10 mM Tris-HCl, pH 8,0, 1 mM EDTA). Se maior concentração de DNA é desejado, o volume pode ser reduzido a 50 mL / poço, mas o rendimento médio total será menor. </li><li> Incubar a placa em temperatura ambiente por um minuto. </li><li> DNA purificado pode ser eluída por qualquer vácuo ou centrifugação. </li><li> Método de vácuo: Coloque placa da coleção clean (DNase, RNase free) no colector de vácuo. Colocar a placa de filtro em cima do colector de vácuo, aplique vácuo de 15 pol Hg (0,5 bar) por 1 minuto até que todos tampão de eluição passou através da placa de DNA de ligação. Coletar DNA purificado (ver Figura 3). </li><li> Método de centrifugação: Coloque placa de purificação do filtro em cima da placa da coleção clean e centrifugar a 1000 xg por 5 minutos. </li></ol> 4. As análises quantitativas e qualitativas <ol><li> Medida OD 260/280, calcular concentração de DNA purificado. </li><li> Realizar análise de gel de agarose do PDNA purificada. </li></ol> 5. Resultados representante E. coli lisado clarificado utilizando centrifugação tradicionais e Pall AcroPrep Adiantamento Placas lisado esclarecimento de filtro mostrou redução significativa de detritos a partir de matéria-prima. (Figura 4). DNA do plasmídeo em três concentrações foi cravado em tampão TE e passado através da placa de esclarecimento AcroPrep Adiantamento lisado filtro. Uma comparação da concentração de DNA plasmidial, pré e pós-filtração, shows ~ recuperação de 100% (Figura 5) em todas as três concentrações. Para demonstrar a recuperação do DNA de E. esclarecidas coli DNA, lisado plasmídeo foi cravado em lisado bruto e, em seguida, esclareceu por filtração. O mesmo DNA do plasmídeo foi adicionado à célula de filtração após lisado. Eletroforese foi realizada em alíquotas de cada amostra (Figura 6). A banda, único e claro de intensidade semelhante de cada amostra demonstra pouco caso de qualquer perda de DNA durante a etapa de clarificação filtração-based. Culturas de E. coli contendo DNA plasmídeo PCAT foram purificados a partir de E. coli lisados ​​usando mL da Pall 350 mL e 1 DNA de ligação placas de filtro, bem como duas outras marcas de 350 placas de DNA mL de ligação. Todas as purificações foram realizados conforme especificado no protocolo recomendado pelo fabricante. A maior concentração de DNA e rendimento total são vistos a partir mL da Pall AcroPrep um avanço placa DNA de purificação do filtro (Figura 7 e Tabela 2). Embora Concorrente W também mostra alta concentração de DNA (155 ng / mL), rendimento de DNA total é a mais baixa (4,7 mg / poço), devido ao volume de recuperação baixo, ~ 30 mL. Concorrente M deu a menor concentração de DNA de 108 ng / mL com um rendimento total de 5,6 mg / poço. Agarose gel análise de DNA purificado PCAT das três placas de DNA diferentes ligação é mostrado na Figura 8. Cada volume da amostra coletada foi ajustado para 65 mL para corrigir diferenças no volume de recuperação antes da eletroforese. Todas as amostras de DNA mostram superenrolado plasmídeo, mas o rendimento do DNA de cada uma das placas de filtro varia. Figura 9 mostra o DNA é tudo preparado na forma superenrolado e tem concentração similar. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Figura 1" /> Figura 1. Montagem de Manifold vácuo de Esclarecimento Lysate. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Figura 2" /> Figura 2. Montagem de Manifold de vácuo para Binding DNA. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Figura 3" /> Figura 3. Montagem de Manifold de vácuo para eluição de vácuo. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Figura 4" /> Figura 4. Filtração simplifica esclarecimento amostra. Triplicatas de 300 mL de lisado bruto clarificado por filtração a vácuo (350 mL AcroPrep Adiantamento placa esclarecimento lisado filtro) ou centrifugação tradicional. OD 320 medido antes e após esclarecimento. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Figura 5" /> Figura 5. Recuperação total dos PDNA após a passagem por Antecipação AcroPrep placa de filtro lisado esclarecimento. 300 mL tampão TE enriquecida com PCAT a 25, 50 e 100 mcg / mL. Concentração de DNA e recuperação calculados a partir de 260 OD antes e após a filtração, N = 2. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Figura 6" /> Figura 6. Recuperação de DNA PCAT após esclarecimento. Quantidades iguais de pUC19 cravado em lisado antes ou depois do esclarecimento, então diluída 1 a 10 em tampão TE. Loaded 2 mL / lane em 1,2% agarose gel. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Figura 7" /> Figura 7. Maior rendimento de DNA plasmidial por poço com pall DNA Avanço Acroprep placa de filtro de ligação de chapas concorrentes. Rendimento DNA PCAT plasmídeo (OD 260) usando placas de purificação de DNA indicou com 1 mL (Pall e M Concorrente) ou 1,5 mL (Concorrente W) cultura overnight de DH5α. Purificação usando o protocolo recomendado pelo fabricante chapa. Barras de erro indicam o erro padrão (n&gt; 6). <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Figura 8" /> Figura 8. Agarose gel análise de DNA purificado PCAT na placa de DNA indicaram vinculativo. 1,2% eletroforese em gel de agarose do PDNA purificada. Amostras combinadas de três purificações separado, eluato ajustada para 65 mL após purificação, 01:10 diluída. Loaded 2 mL por pista. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Figura 9" /> Figura 9. Qualidade e de recuperação do DNA puro que quando os métodos de centrifugação ou a vácuo são usados ​​para a final eluato eletroforese em gel de agarose coleção do PDNA purificada. Protocolo recomendado pelo fabricante de placas Adiantamento AcroPrep filtro foi seguido para a purificação PDNA. Eluição foi realizada por centrifugação a 1.000 xg por 5 minutos ou a vácuo com 15 pol Hg (0,5 bar) por 2 minutos.

Os autores deste artigo de vídeo-são funcionários da Pall Life Sciences que produzem os instrumentos e reagentes apresentado no vídeo e texto.

Nós descrevemos um protocolo, facile simplificada de placas de filtro AcroPrep antemão que oferece várias vantagens. Este método oferece rendimentos máximos DNA plasmídeo quando processados ​​em formatos Avanço manual, semi-automática ou totalmente automatizadas AcroPrep. Um pré-filtro integrada na placa de depuração lisado fornece filtração consistentes de amostras, inclusive aqueles contendo altos níveis de partículas. A geometria dos poços da placa de resultados no mais rápido, taxas de filtração mais uniforme em toda a placa com o volume mínimo de hold-up, ea geometria da ponta de saída fornece fluxo direto de amostras em placa de receptor sem preocupações de contaminação cruzada. Importante, a membrana de alta capacidade de ligação em chapa de DNA de ligação de placas Adiantamento AcroPrep filtro é otimizado para recuperação máxima de DNA de alta qualidade plasmídeo.

<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

Descrevemos o processo completo de Placas Adiantamento AcroPrep Filtro para 96 ​​preparações plasmídeo, a partir de cultura procarióticas e terminando com DNA de alta pureza. Baseado em multi-filtração para o apuramento bem lisado bacteriano e purificação de DNA, esse método cria um processo simplificado para a preparação de plasmídeo. Placas de filtro à base de sílica contendo mídia pode facilmente ser processado por filtração a vácuo ou centrifugação para produzir quantidades apreciáveis ​​de DNA plasmidial. Análises quantitativas determinar o DNA purificado plasmídeo é consistentemente de alta qualidade com média de OD 260/280 índices de 1,97. No geral, os rendimentos plasmídeo oferecer DNA mais puro para aplicações a jusante, tais como seqüenciamento e clonagem. Este método simplificado de usar placas de Adiantamento AcroPrep filtro permite o processamento manual, semi-automática ou totalmente automatizadas.

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

Este protocolo é uma alternativa rentável para o esclarecimento paralela eficiente e purificação de DNA plasmidial de<em&gt; E. coli</em&gt; Culturas. O processo de Adiantamento AcroPrep começa com uma placa de filtro otimizado lisado esclarecimento seguida de purificação em uma ligação de alta capacidade de ligação DNA placa do filtro.

Purificação simplificada do DNA plasmidial das culturas Procarióticas

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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Vídeo: 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.

Uma Abordagem DNA afinidade G-quadruplex para a purificação de enzimaticamente activa 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 ...

Bioquímica

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.

Purificação de DNA plasmidial colônias bacterianas Usando o Kit Qiagen Miniprep

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

Biologia

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.

Uma purificação e ensaio de atividade In Vitro para um ppGpp (p) sintetase de Clostridium difficile

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

Imunologia e Infecção

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.

De alto rendimento de purificação de afinidade com tag Proteínas Recombinantes

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.

Ensaios Repórter Massively Parallel em células de mamíferos cultivadas

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.

Análise de estabilidade de plasmídeo com microfluídica de gotículas de código aberto

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

Bioengenharia

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 %.

Uso de montagem in vivo para construção de plasmídeo de alta eficiência

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.

Strand DNA Deslocamento Portas para redes de reação de Execução químicos derivados do plasmídeo

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.

A purificação de todo o genoma de DNA circular Extracromossômica de células eucarióticas

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: montagem de quimera por recuperação de plasmídeo e inserção de sítio de enzima de restrição

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