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. Liquidación lisado <ol><li> Crecer E. culturas coli transformadas con el ADN de plásmido deseado en profundidad placas (1 ml de la cultura / pocillo) en caldo Luria con los antibióticos apropiados durante la noche a 37 ° C con agitación. </li><li> Pellet E. coli en la placa de cultivo a 5.000 xg durante 10 minutos, luego decantar el sobrenadante. </li><li> Resuspender cada pellet en 100 l de búfer Resuspensión (50 mM Tris-HCl, pH 8,0, 10 mM EDTA, 100 mg / ml RNasa A). </li><li> Añadir 100 tampón de lisis l / pocillo (200 mM NaOH, 1% SDS) y agitar con agitador de placas durante 2 minutos. </li><li> Añadir 100 l / pocillo de búfer neutralización (3,0 M de acetato de potasio, pH 5,5) y agitar durante 2 minutos. </li><li> Transferencia de lisado de células de la placa de lisado aclaración. </li><li> Coloque la placa de unión al ADN en la parte superior de la placa de recolección de 350 l, y el lugar en el fondo del colector de vacío. Montar colector de vacío (ver Figura 1). </li><li> Lisado lugar aclarar la placa en la parte superior de los aparatos de vacío. </li><li> Aplicar un vacío de 10 pulgadas de mercurio (0,34 bar) y recoger filtrado en la placa de unión al ADN. No utilice vacío superior a 12 mm Hg. (0,41 bar). </li><li> Desmontar colector de vacío. Coloque 2 ml de residuos recogida en la placa inferior del aparato. </li></ol> 2. De unión con ADN <ol><li> Mover la placa de unión del ADN a la parte superior de la placa (ver Figura 2). </li><li> Añadir 300 l Buffer / Encuadernación y (6 M guanidina-HCl) y una pipeta de arriba a abajo para mezclar. </li><li> Aplicar un vacío [~ 5 mm Hg (0,17 bar) para el vacío lento] y desechar filtrado. ADN está ligado a la membrana. </li><li> Lavar con 400 l / pocillo de tampón de lavado (80% de etanol). </li><li> Aplicar un vacío, a continuación, descartar el filtrado. Deseche innecesaria si se usa 2 colecta ml o filtrando directamente a la basura. </li><li> Repetir el lavado una vez. </li><li> Blot parte inferior de la placa de filtro sobre una toalla absorbente para secar. </li><li> Aplicar un vacío, una vez más durante 5-10 minutos para asegurar la eliminación de los restos de alcohol. </li><li> Blot fondo para asegurar la eliminación de las gotas de etanol. </li></ol> 3. Elución del ADN <ol><li> Añadir 70 l / y tampón de elución (10 mM Tris-HCl, pH 8,0, 1 mM EDTA). Si la mayor concentración de ADN que se desea, el volumen se reduce a 50 l / bien, pero el rendimiento total promedio será menor. </li><li> Incubar la placa a temperatura ambiente durante un minuto. </li><li> ADN purificado se eluye ya sea por vacío o centrifugación. </li><li> Método de vacío: Coloque la placa de recolección de limpia (DNasa, RNasa libre) en el colector de vacío. Colocar la placa de filtro en la parte superior del colector de vacío, aplicar vacío a 15 pulgadas de mercurio (0,5 bar) durante 1 minuto hasta que todos los buffer de elución ha pasado a través de la placa de unión al ADN. Recoger ADN purificado (ver Figura 3). </li><li> Método de centrifugación: Colocar la placa de purificación de filtro en la parte superior de la placa de recolección de limpia y se centrifuga a 1000 xg durante 5 minutos. </li></ol> 4. Los análisis cuantitativos y cualitativos <ol><li> Medida de OD 260/280, calcular la concentración de ADN purificado. </li><li> Realizar un análisis en gel de agarosa de la pDNA purificada. </li></ol> 5. Resultados representante E. coli lisado clarificado mediante centrifugación tradicional y Pall AcroPrep adelantado placas aclarar lisado filtro mostraron una reducción apreciable de los desechos de material de partida. (Figura 4). ADN plásmido en tres concentraciones, se añadieron en buffer TE y pasa a través de la placa de Avance AcroPrep aclarar lisado filtro. Una comparación de la concentración de ADN plásmido, antes y después de la filtración, muestra a 100% de recuperación (Figura 5) en las tres concentraciones. Para demostrar la recuperación de ADN de aclarar E. coli lisado ADN plásmido, se añadieron en lisado crudo y se clarificarán por filtración. El ADN del plásmido mismo se añadió a la filtración después de lisado celular. La electroforesis se realizó en alícuotas de cada muestra (Figura 6). Una banda única y clara de similar intensidad en cada ejemplo muestra poca o ninguna pérdida de ADN durante la fase de clarificación y filtración basados. Los cultivos de E. coli que contiene el ADN del plásmido pCAT fueron purificados de E. coli lisados ​​con 350 l de Pall y 1 ml de ADN filtro de placas de unión, así como otras dos marcas de 350 placas de unión al ADN l. Todas las purificaciones se realizaron según lo especificado en el protocolo recomendado por el fabricante. La mayor concentración de ADN y la producción total se ven desde un Pall ml adelantado AcroPrep ADN de purificación de placas de filtro (Figura 7 y Tabla 2). A pesar de la competencia W también muestra la concentración de ADN de alto (155 ng / l), el rendimiento total de ADN es la más baja (4,7 mg / así) debido a la recuperación del volumen bajo, aproximadamente 30 mL. Competidor M dio la menor concentración de ADN a 108 ng / l con un rendimiento total de 5,6 ug / pocillo. Agarose análisis en gel de ADN purificado pCAT de las tres placas de unión al ADN diferente se muestra en la Figura 8. Cada volumen de la muestra recogida se ajustó a 65 l para corregir las diferencias en el volumen de recuperación antes de la electroforesis. Todas las muestras de ADN de plásmido superenrollado mostrar, pero varía el rendimiento de ADN de cada una de las placas de filtro. La Figura 9 muestra que el ADN es preparado en forma de superenrollado y tiene una concentración similar. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Figura 1" /> Figura 1. Asamblea del colector de vacío para el Esclarecimiento lisado. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Figura 2" /> Figura 2. Asamblea del colector de vacío para la unión de ADN. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Figura 3" /> Figura 3. Asamblea del colector de vacío para la elución de vacío. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Figura 4" /> Figura 4. Filtración simplifica la aclaración de la muestra. Tres muestras de 300 l de lisado crudo aclaró por filtración al vacío (350 l Avance AcroPrep lisado placa aclaración filtro) o centrifugación tradicional. OD 320 antes y después de la aclaración. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Figura 5" /> Figura 5. La recuperación total de pDNA tras el paso por adelantado AcroPrep placa de filtro lisado aclaración. 300 L de buffer TE enriquecida con pCAT a 25, 50 y 100 ug / mL. Concentración de ADN y la recuperación calculado a partir de 260 OD antes y después de la filtración, N = 2. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Figura 6" /> Figura 6. La recuperación de ADN pCAT después de la clarificación. Igual cantidad de pUC19 añadidos en lisado antes o después de la aclaración, entonces se diluye 1 a 10 en buffer TE. Cargado 2 l / carril en el 1,2% en gel de agarosa. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Figura 7" /> Figura 7. Mayor rendimiento de ADN plasmídico por pocillo con ADN manto adelantado Acroprep placa de unión que las placas de filtro de la competencia. PCAT rendimiento de ADN plásmido (OD 260) se indica con placas de purificación de ADN con 1 ml (Pall M y la competencia) o 1,5 mL (Competidor W) durante la noche de la cultura DH5a. Purificación mediante el protocolo recomendado por el fabricante de la placa. Las barras de error indican el error estándar (n&gt; 6). <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Figura 8" /> Figura 8. Agarosa análisis en gel de ADN purificado pCAT en la placa de unión al ADN indicó. Agarosa al 1,2% electroforesis en gel de la pDNA purificada. Muestras combinadas de las tres purificaciones por separado, eluido ajustado a 65 l después de la purificación, diluido 1:10. Cargado 2 l por carril. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Figura 9" /> Figura 9. La calidad y la recuperación de ADN puro similares cuando los métodos de centrifugación o vacío se utilizan para la final de la electroforesis en gel de agarosa colección eluido de la pDNA purificada. Protocolo recomendado por el fabricante para el Avance AcroPrep filtro de placas se ha seguido para la purificación pDNA. La elución se realizó por centrifugación a 1.000 xg durante 5 minutos o una aspiradora con 15 pulgadas de mercurio (0,5 bar) durante 2 minutos.

Los autores de este video-artículo son empleados de Pall Life Sciences que producen los instrumentos y reactivos aparecen en el video y el texto.

Se describe un protocolo fácil, ágil de las placas de filtro AcroPrep anticipada que ofrece varias ventajas. Este método proporciona un rendimiento máximo de ADN plásmido cuando se procesan en formatos de avance manual, semiautomático o totalmente automatizado-AcroPrep. Un pre-filtro integrado en la placa de autorización lisado proporciona una filtración de las muestras, incluso las que contengan altos de partículas. La geometría y de los resultados de la placa más rápida, las tasas de filtración más uniforme a través de la placa con un mínimo de volumen de retención, y la geometría de la punta de salida proporciona un flujo directo de muestras en el plato receptor sin problemas de contaminación cruzada. Es importante destacar que la membrana de alta capacidad de unión de la placa de unión al ADN de AcroPrep adelantado placas de filtro está optimizado para una máxima recuperación de la calidad del ADN plásmido de alto.

<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

Se describe el proceso completo de placas adelantado AcroPrep de filtro para 96 ​​preparaciones de plásmidos, a partir de la cultura procariotas y terminando con el ADN de alta pureza. Sobre la base de múltiples pozos de filtración para el despacho de lisado bacteriano y purificación del ADN, este método crea un proceso simplificado para la preparación de plásmido. Placas de filtro que contiene los medios de comunicación a base de sílice pueden ser fácilmente transformados por filtración al vacío o centrifugación para producir cantidades apreciables de ADN del plásmido. Los análisis cuantitativos determinar el ADN de plásmido purificado es siempre de alta calidad con un promedio de OD 260/280 ratios de 1,97. En general, los rendimientos de plásmido ADN ofrecen más puro para aplicaciones posteriores, como la secuenciación y clonación. Este método simplificado de la utilización de placas AcroPrep Avance del filtro permite el procesamiento manual, semiautomático o completamente automático.

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 es una alternativa rentable para la clarificación eficiente en paralelo y purificación del ADN plasmídico a partir de<em&gt; E. coli</em&gt; Culturas. El proceso de avance AcroPrep comienza con una placa de lisado aclaración optimizado filtro seguido por la purificación en una alta capacidad de unión al ADN vinculante placa de filtro.

Purificación simplificada de ADN plásmido de las Culturas procariotas

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

streamlined-purification-of-plasmid-dna-from-prokaryotic-cultures

Research

JoVE Journal

Biology

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Video: 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.

Un enfoque ADN afinidad G-cuádruplex para la purificación de enzimáticamente 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 ...

Investigación

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.

Purificación del ADN plásmido de las colonias de bacterias Utilizando el kit de 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 ...

Biología

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.

Una purificación y análisis de actividad In Vitro de una (p) ppGpp sintetasa de Clostridium difficile

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

Inmunología e Infección

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 rendimiento de purificación de afinidad de proteínas marcadas con 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 ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Ingeniería de agentes antivirales a través de la resonancia de plasmones de superficie

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

Bioingeniería

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.

Análisis de estabilidad de plásmidos con microfluídica de gotas de código abierto

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

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 ensamblaje in vivo para la construcción de plásmidos de alta eficiencia

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.

Plásmido derivado de ADN Strand Displacement Puertas de Redes de reacción de Ejecución Química

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.

La purificación de todo el genoma de ADN circular Extracromosómica a partir de células eucariotas

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: Ensamblaje de quimeras por inserción de sitios de enzimas de recuperación y restricción de plásmidos

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