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. Dégagement Lysat <ol><li> Cultivez E. cultures coli transformée avec l&#39;ADN plasmidique désiré dans assiettes creuses bien (1 ml de culture / puits) dans le bouillon Luria avec des antibiotiques appropriés nuit à 37 ° C avec agitation. </li><li> Pellet E. coli dans des plaques de culture à 5000 xg pendant 10 minutes, puis le surnageant décanter. </li><li> Resuspendre chaque pastille dans 100 uL tampon de resuspension (50 mM Tris-HCI, pH 8,0, EDTA 10 mM, 100 ug / ml de RNase A). </li><li> Ajouter 100 Lysis Buffer ul / puits (200 mM NaOH, 1% SDS) et agiter à l&#39;aide Agitateur de plaque pendant 2 minutes. </li><li> Ajouter 100 tampon de neutralisation ul / puits (3,0 M acétate de potassium, pH 5,5) et agiter pendant 2 minutes. </li><li> Transfert lysat cellulaire à la plaque de lysat de clarification. </li><li> Plaque d&#39;ADN Placez contraignante sur le dessus de 350 uL plaque de collection et le lieu dans le fond du collecteur à vide. Assemblez collecteur à vide (voir Figure 1). </li><li> Plaque de la Place des éclaircissements sur le dessus du lysat appareil à vide. </li><li> Appliquer vide à 10 po Hg (0,34 bar) et recueillir filtrat dans l&#39;ADN plaque de fixation. Ne pas utiliser à vide supérieure à 12 po Hg. (0,41 bar). </li><li> Démonter collecteur à vide. Placer 2 ml plaque de collection des déchets dans le fond de l&#39;appareil. </li></ol> 2. Liaison à l&#39;ADN <ol><li> Déplacer la plaque de fixation d&#39;ADN au sommet du collecteur (voir Figure 2). </li><li> Ajouter 300 pi de tampon / puits reliure (6 M guanidine-HCl) et une pipette de haut en bas pour mélanger. </li><li> Appliquer le vide [~ 5 po Hg (0,17 bar) pour le vide lente] et jetez filtrat. L&#39;ADN est maintenant lié à la membrane. </li><li> Laver avec 400 ul / puits de tampon de lavage (80% d&#39;éthanol). </li><li> Appliquer le vide, puis jeter le filtrat. Jeter inutiles si vous utilisez deux plaques de collection ml ou de filtrage directement sur les déchets. </li><li> Répétez laver une fois. </li><li> Blot en bas de la plaque de filtre sur une serviette absorbante pour sécher. </li><li> Appliquer une fois de plus à vide pendant 5-10 minutes pour assurer l&#39;élimination de l&#39;alcool résiduel. </li><li> Blot en bas pour assurer l&#39;élimination de gouttelettes d&#39;éthanol. </li></ol> 3. Élution d&#39;ADN <ol><li> Ajouter 70 Elution Buffer ul / puits (10 mM Tris-HCI, pH 8,0, EDTA 1 mM). Si la concentration élevée d&#39;ADN est désiré, le volume peut être réduit à 50 pl / puits, mais le rendement moyen total sera inférieur. </li><li> Incuber la plaque à température ambiante pendant une minute. </li><li> ADN purifié peut être élue par soit vide ou centrifugation. </li><li> Méthode sous vide: Placer la plaque de collecte propre (DNase, RNase) dans le collecteur d&#39;aspiration. Placez la plaque de filtre sur le dessus du collecteur à vide, appliquer le vide à 15 po Hg (0,5 bar) pendant 1 minute jusqu&#39;à ce que tous tampon d&#39;élution a passé à travers la plaque de fixation d&#39;ADN. Recueillir l&#39;ADN purifié (voir Figure 3). </li><li> Méthode de centrifugation: plaque de purification de la Place de filtre sur le dessus de plaque de collection propre et centrifuger à 1000 xg pendant 5 minutes. </li></ol> 4. Analyses qualitative et quantitative <ol><li> Mesure OD 260/280, calculer la concentration d&#39;ADN purifié. </li><li> Effectuer une analyse sur gel d&#39;agarose de l&#39;ADNp purifiée. </li></ol> 5. Les résultats représentatifs E. coli lysat clarifié par centrifugation traditionnelle et Pall AcroPrep Plaques Advance lysat éclaircissements Filtre a montré une réduction appréciable de débris de matériau de départ. (Figure 4). L&#39;ADN plasmidique à trois concentrations a été enrichi en tampon TE et passé à travers la plaque Advance AcroPrep clarification du lysat filtre. Une comparaison de la concentration de l&#39;ADN plasmidique, pré-et post-filtration, montre ~ récupération de 100% (figure 5) à tous les trois concentrations. Pour démontrer la récupération de l&#39;ADN de clarifier E. coli lysat, l&#39;ADN plasmidique a été enrichi en lysat brut et ensuite clarifié par filtration. L&#39;ADN plasmidique mêmes a été ajouté à filtration après lysat cellulaire. L&#39;électrophorèse a été réalisée sur des aliquotes de chaque échantillon (figure 6). Une seule bande claire d&#39;intensité similaire à partir de chaque échantillon démontre peu ou pas de perte de l&#39;ADN lors de l&#39;étape de clarification de filtration à base. Les cultures de E. coli contenant l&#39;ADN plasmidique PCAT ont été purifiés à partir de E. coli lysats utilisant 350 uL de Pall et 1 ml liaison à l&#39;ADN des plaques de filtration, ainsi que deux autres marques de 350 plaques d&#39;ADN uL contraignant. Tous les purifications ont été effectuées comme spécifié dans le protocole recommandé par le fabricant. La plus forte concentration de l&#39;ADN et le rendement total sont vus de Pall 1 ml Advance AcroPrep purification ADN filtre à plaques (figure 7 et tableau 2). Bien Compétiteur W montre également la concentration d&#39;ADN élevée (155 ng / uL), le rendement total de l&#39;ADN est le plus bas (4,7 ug / puits) en raison du volume faible récupération, ~ 30 uL. Concurrent M a donné la plus faible concentration de l&#39;ADN à 108 ng / uL avec un rendement total de 5,6 ug / puits. Agarose gel de l&#39;analyse d&#39;ADN purifié à partir PCAT les trois plaques d&#39;ADN différentes de liaison est montré dans la figure 8. Chaque volume de l&#39;échantillon recueilli a été ajusté à 65 uL de corriger les différences de volume de récupération avant l&#39;électrophorèse. Tous les échantillons montrent superenroulé ADN plasmidique, mais le rendement d&#39;ADN de chacune des plaques de filtration varie. La figure 9 montre l&#39;ADN préparé tout est dans la forme et a superenroulé concentration similaire. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Figure 1" /> Figure 1. Assemblée du collecteur d&#39;aspiration pour clarification du lysat. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Figure 2" /> Figure 2. Assemblée du collecteur d&#39;aspiration pour fixation à l&#39;ADN. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Figure 3" /> Figure 3. Assemblée des Collecteur à vide pour l&#39;élution à vide. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Figure 4" /> Figure 4. Filtration simplifie la clarification de l&#39;échantillon. Trois échantillons de 300 ul de lysat brut clarifiée par filtration sous vide (350 uL Advance AcroPrep clarification du lysat filtre à plaques) ou la centrifugation traditionnelle. OD 320 mesurés avant et après clarification. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Figure 5" /> Figure 5. Le rétablissement complet de la ADNp après passage à travers la plaque Advance AcroPrep lysat éclaircissements filtre. 300 pi de tampon TE dopés avec PCAT à 25, 50 et 100 pg / mL. Concentration de l&#39;ADN et la récupération calculée à partir de 260 OD avant et après filtration, N = 2. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Figure 6" /> Figure 6. Récupération de l&#39;ADN PCAT après clarification. Des quantités égales de pUC19 pointes en lysat avant ou après la clarification, puis diluée 1 à 10 dans du tampon TE. Chargée 2 pl / voies sur 1,2% gel d&#39;agarose. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Figure 7" /> Figure 7. Rendement plus élevé d&#39;ADN plasmidique par puits avec de l&#39;ADN Pall Advance AcroPrep contraignante plaque filtrante que les plaques concurrent. PCAT le rendement d&#39;ADN plasmidique (DO 260) en utilisant des plaques de purification d&#39;ADN indiquée avec 1 ml (Pall et M concurrent) ou 1,5 ml (Compétiteur W) culture de nuit de DH5a. Purification utilisant le protocole recommandé par le fabricant plaque. Les barres d&#39;erreur indiquent l&#39;erreur standard (n&gt; 6). <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Figure 8" /> Figure 8. Gel d&#39;agarose analyse de l&#39;ADN purifié sur plaque de PCAT ADN indique contraignant. 1,2% électrophorèse sur gel d&#39;agarose des ADNp purifiée. Échantillons groupés par trois purifications séparées, éluat ajusté à 65 uL après purification, dilué à 1:10. Chargée 2 uL par voie. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Figure 9" /> Figure 9. La qualité et la récupération d&#39;ADN pur similaires lorsque les méthodes de centrifugation ou sous vide sont utilisés pour la collecte d&#39;électrophorèse sur gel d&#39;agarose éluat final de l&#39;ADNp purifiée. Protocole recommandé par le fabricant pour les plaques AcroPrep Filtre Advance a été suivie pour la purification ADNp. L&#39;élution a été effectuée par centrifugation à 1000 xg pendant 5 minutes ou passer l&#39;aspirateur avec 15 po Hg (0,5 bar) pendant 2 minutes.

Les auteurs de cette vidéo-article sont des employés de Pall Life Sciences qui produisent de l&#39;instrument et des réactifs en vedette dans la vidéo et du texte.

Nous décrivons une faciles, protocole simplifié de Plaques AcroPrep Filtre Advance qui offre plusieurs avantages. Cette méthode offre un maximum de rendements d&#39;ADN plasmidique lorsqu&#39;il est traité dans le manuel, semi-automatiques ou entièrement automatisés formats Advance AcroPrep. Un préfiltre intégré sur la plaque de dégagement lysat assure la filtration uniforme des échantillons, même ceux contenant des teneurs élevées de particules. La géométrie ainsi des résultats plus rapides dans les plaques, les taux de filtration plus uniforme à travers la plaque avec un minimum de volume de rétention, et la géométrie de la pointe de sortie fournit des flux directs d&#39;échantillons dans la plaque du récepteur sans préoccupations de contamination croisée. Surtout, la membrane à haute capacité obligatoire dans la plaque de fixation d&#39;ADN des plaques AcroPrep Filtre Advance est optimisé pour une récupération maximale de l&#39;ADN plasmidique de haute qualité.

<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

Nous décrivons le processus complet de plaques Advance AcroPrep filtre pour 96 préparations plasmidiques, à partir de la culture procaryotes et se terminant avec l&#39;ADN de haute pureté. Basé sur plusieurs puits de filtration pour le dédouanement lysat bactérien et la purification de l&#39;ADN, cette méthode crée un processus simplifié pour la préparation de plasmide. Filtre plaques contenant de la silice les médias peuvent être facilement traitées par filtration sous vide ou une centrifugeuse pour produire des quantités appréciables de l&#39;ADN plasmidique. Les analyses quantitatives de déterminer l&#39;ADN plasmide purifié est constamment de haute qualité avec DO moyenne 260/280 ratios de 1,97. Globalement, les rendements plasmide ADN offrent plus pur pour des applications en aval, telles que le séquençage et le clonage. Cette méthode simplifiée de l&#39;aide de plaques Advance AcroPrep filtre permet un traitement manuel, semi-automatique ou entièrement automatique.

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

Ce protocole est une alternative rentable pour les éclaircissements parallèles efficaces et de purification d&#39;ADN plasmidique à partir de<em&gt; E. coli</em&gt; Cultures. Le processus avance AcroPrep commence avec une plaque de clarification du lysat optimisé filtre suivi d&#39;une purification sur l&#39;ADN de haute capacité de liaison liant plaque filtrante.

Purification simplifiée d&#39;ADN plasmidique de cultures procaryotes

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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Vidéo: 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.

Une approche du G-quadruplex ADN-affinité pour la purification de l&#39;activité enzymatique 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 ...

Recherche

Biochimie

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.

Purifiant l&#39;ADN plasmidique des colonies bactériennes en utilisant le kit 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 ...

Biologie

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.

Une Purification et analyse d’activité In Vitro pour un (p) ppGpp synthétase de Clostridium difficile

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

Immunologie et infection

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.

À haut débit Affinity Purification de protéines recombinantes-étiquette

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.

Essais Reporter massivement parallèles dans des cellules de mammifères en culture

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.

Analyse de la stabilité des plasmides avec la microfluidique de gouttelettes open source

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

Bio-ingénierie

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 %.

Utilisation de l’assemblage in vivo pour la construction de plasmides à haut rendement

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.

Plasmide dérivé d&#39;ADN Strand Displacement Gates Réseaux de réaction d&#39;exécution chimiques

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.

Purification de l&#39;ensemble du génome de l&#39;ADN extrachromosomique circulaire à partir de cellules eucaryotes

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: Assemblage de la chimère par insertion de plasmides et 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 ...

Purification simplifiée d'ADN plasmidique de cultures procaryotes

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