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. Liquidazione lisato <ol><li> Crescere E. culture coli trasformato con DNA plasmidico desiderato in profondo piastre (1 ml di coltura / pozzetto) in brodo Luria con antibiotico appropriato overnight a 37 ° C con agitazione. </li><li> Pellet di E. coli in piastra di coltura a 5.000 xg per 10 minuti, poi decantare il surnatante. </li><li> Risospendere ogni pellet in 100 microlitri Resuspension Buffer (50 mM Tris-HCl, pH 8,0, 10 mM EDTA, 100 mg / ml RNasi A). </li><li> Aggiungere 100 microlitri Lysis Buffer / e (200 mM NaOH, 1% SDS) e agitare utilizzando con agitazione per 2 minuti. </li><li> Aggiungere 100 l / pozzetto tampone di neutralizzazione (3,0 M acetato di potassio, pH 5,5) e agitare per 2 minuti. </li><li> Trasferimento lisato cellulare di lisato piastra chiarimenti. </li><li> Inserire la piastra di DNA vincolante sulla parte superiore di 350 microlitri e piastra di raccolta posto sul fondo del collettore di aspirazione. Assemblare collettore di vuoto (vedi Figura 1). </li><li> Luogo chiarimenti piastra lisato sulla parte superiore del dispositivo vuoto. </li><li> Applicare il vuoto a 10 pollici Hg (0,34 bar) e raccogliere filtrato nella piastra di legame al DNA. Non utilizzare a vuoto superiore a 12 in Hg. (0,41 bar). </li><li> Smontare collettore di aspirazione. Mettere 2 ml di raccolta dei rifiuti nella piastra inferiore di apparecchi. </li></ol> 2. DNA Binding <ol><li> Spostare la piastra di legame del DNA alla parte superiore del collettore (vedi figura 2). </li><li> Aggiungere 300 microlitri Buffer / e Binding (6 M guanidina-HCl) e pipettare su e giù per mescolare. </li><li> Applicare il vuoto [~ 5 pollici Hg (0,17 bar) per il vuoto lenta] e scartare filtrato. Il DNA è ora associato alla membrana. </li><li> Lavare con 400 microlitri / pozzetto di soluzione di lavaggio (80% etanolo). </li><li> Applicare il vuoto, poi scarta il filtrato. Eliminare inutili se si usa 2 Piastra raccolta ml o filtrando direttamente da perdere. </li><li> Ripetere il lavaggio una volta. </li><li> Fondo macchia di piastra di filtraggio sul tovagliolo assorbente ad asciugare. </li><li> Applicare il vuoto ancora una volta per 5-10 minuti per assicurare la rimozione di residui di alcool. </li><li> Blot fondo per assicurare la rimozione di goccioline etanolo. </li></ol> 3. Eluizione del DNA <ol><li> Aggiungere 70 microlitri / e tampone di eluizione (10 mM Tris-HCl, pH 8,0, 1 mM EDTA). Se la concentrazione di DNA si desidera più, il volume può essere ridotto a 50 microlitri / bene, ma la resa media totale sarà inferiore. </li><li> Incubare la piastra a temperatura ambiente per un minuto. </li><li> DNA purificato può essere eluito sia vuoto o centrifugazione. </li><li> Metodo vuoto: Inserire la piastra di raccolta pulita (DNasi, RNasi free) nel collettore di aspirazione. Piastra posto del filtro sulla parte superiore del collettore di aspirazione, applicare vuoto a 15 pollici Hg (0,5 bar) per 1 minuto fino a quando tutti i buffer di eluizione è passato attraverso la piastra di legame al DNA. Raccogliere DNA purificato (vedi figura 3). </li><li> Metodo di centrifugazione: purificazione piastra Luogo filtro sulla parte superiore della piastra di raccolta pulita e centrifugare a 1000 xg per 5 minuti. </li></ol> 4. Analisi quantitativa e qualitativa <ol><li> Misura 260/280 OD, calcolare la concentrazione di DNA purificato. </li><li> Effettuare analisi di gel di agarosio del PDNA purificato. </li></ol> 5. Rappresentante Risultati E. coli lisato chiarificato mediante centrifugazione tradizionali e Pall AcroPrep Advance Piastre chiarimenti lisato Filtro mostrato sensibile riduzione di detriti da materiale di partenza. (Figura 4). DNA plasmidico a tre concentrazioni è stata aggiunta in TE buffer e passato attraverso la piastra di chiarificazione Advance AcroPrep lisato filtro. Un confronto tra concentrazione di DNA plasmide, pre-e post-filtrazione, mostra ~ 100% di recupero (Figura 5) in tutti e tre concentrazioni. Per dimostrare il recupero di DNA da E. chiarito coli lisato, il DNA plasmidico è stata aggiunta in lisato grezzo e successivamente chiarificati mediante filtrazione. Lo stesso DNA plasmidico è stato aggiunto alla filtrazione seguenti lisato cellulare. Elettroforesi è stata eseguita su aliquote di ciascun campione (Figura 6). Un singolo, banda chiara di intensità simile da ciascun campione dimostra poca o nessuna perdita di DNA durante la filtrazione basata fase di chiarificazione. Culture di E. coli contenente DNA plasmidico pCAT sono state purificate da E. coli lisati con Pall 350 microlitri e 1 ml di DNA Piastre vincolante filtro, così come altri due marchi di 350 piastre DNA microlitri vincolanti. Tutte le purificazioni sono state eseguite come specificato nel protocollo raccomandato dal produttore. La più alta concentrazione di DNA e di resa totale sono visti da Pall 1 Anticipo ml DNA piastra AcroPrep purificazione filtro (Figura 7 e Tabella 2). Sebbene concorrente W mostra anche ad alta concentrazione di DNA (155 ng / mL), la resa del DNA totale è il più basso (4,7 mg / pozzetto) a causa del volume di ripristino bassa, ~ 30 microlitri. Concorrente M ha dato la più bassa concentrazione di DNA in 108 ng / mL, con un rendimento totale di 5,6 mcg / bene. Agarose analisi gel di DNA purificato pCAT dalle tre piastre DNA diversi vincolante è mostrato nella Figura 8. Ogni volume campione raccolto è stato rettificato a 65 microlitri di correggere le differenze nel volume di ripristino prima di elettroforesi. Tutti i campioni mostrano superavvolto DNA plasmidico, ma resa DNA da ciascuna delle piastre del filtro varia. La figura 9 mostra il DNA preparato è tutto in forma e ha superavvolto concentrazione simile. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Figura 1" /> Figura 1. Assemblea dei Collettore sottovuoto per il chiarimento lisato. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Figura 2" /> Figura 2. Assemblea dei Collettore sottovuoto per l&#39;associazione del DNA. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Figura 3" /> Figura 3. Assemblea dei Collettore sottovuoto per l&#39;eluizione sotto vuoto. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Figura 4" /> Figura 4. Filtrazione semplifica la chiarificazione del campione. Campioni triplice copia di 300 ml di lisato grezzo chiarito filtrazione sotto vuoto (350 microlitri Advance AcroPrep piastra lisato chiarimenti filtro) o centrifugazione tradizionali. OD 320 misurati prima e dopo il chiarimento. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Figura 5" /> Figura 5. Pieno recupero di PDNA dopo passaggio piastra Advance filtro AcroPrep lisato chiarimenti. 300 microlitri di buffer TE spillo con pCAT a 25, 50 e 100 mg / ml. Concentrazione di DNA e di recupero può essere calcolato da OD 260 prima e dopo la filtrazione, N = 2. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Figura 6" /> Figura 6. Recupero del DNA pCAT dopo chiarimenti. La stessa quantità di pUC19 spillo in lisato prima o dopo la precisazione, poi diluito 1 a 10 in tampone TE. Caricato 2 microlitri / corsia su gel di agarosio 1,2%. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Figura 7" /> Figura 7. Maggiore resa DNA plasmidico per bene con il DNA pall Advance AcroPrep vincolante piatto filtro rispetto alle piastre concorrente. PCAT resa DNA plasmidico (OD 260) con indicato piatti purificazione del DNA con 1 ml (Pall e M concorrente) o 1,5 ml (concorrente W) di ​​cultura durante la notte DH5α. Purificazione mediante protocollo raccomandato produttore piastra. Barre di errore indicano l&#39;errore standard (n&gt; 6). <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Figura 8" /> Figura 8. Gel di agarosio analisi del DNA purificato pCAT sulla piastra del DNA indicato vincolanti. 1,2% gel elettroforesi di agarosio del PDNA purificato. Campioni riuniti da tre purificazioni separati, eluato regolata a 65 microlitri dopo la depurazione, diluito 1:10. Caricato 2 microlitri per corsia. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Figura 9" /> Figura 9. Qualità e il recupero di dna puro simili quando i metodi centrifuga o per vuoto vengono utilizzati per la raccolta finale elettroforesi gel agarosio eluato del PDNA purificato. Protocollo raccomandato dal produttore per Piastre Advance AcroPrep filtro è stato seguito per la purificazione PDNA. Eluizione è stata eseguita da centrifugare a 1000 xg per 5 minuti o per vuoto con 15 pollici Hg (0,5 bar) per 2 minuti.

Gli autori di questo video-articolo sono dipendenti di Pall Life Sciences che producono lo strumento e reagenti presenti nel video e testo.

Descriviamo un facile, protocollo semplificato di Piastre Advance Filter AcroPrep che offre diversi vantaggi. Questo metodo fornisce la massima produzione di DNA plasmidico quando trattati in formati AcroPrep manuale, semiautomatica o completamente automatica Advance. Un pre-filtro integrato sulla piastra spazio lisato fornisce la filtrazione costante di campioni, anche quelli contenenti livelli elevati di particolato. La geometria e dei risultati piatto più veloce, i tassi di filtrazione più uniforme in tutto il piatto con il minimo volume di hold-up, e la geometria della punta di uscita fornisce un flusso diretto di campioni in piastra ricevitore senza preoccupazioni di contaminazione incrociata. È importante sottolineare che la membrana ad alta capacità di legame nel piatto legame del DNA di Piastre Advance AcroPrep filtro è ottimizzato per il recupero massimo di DNA di alta qualità plasmide.

<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

Noi descriviamo il processo completo di piastre Advance AcroPrep filtro per 96 preparazioni plasmide, a partire dalla cultura procariotiche e termina con il DNA ad alta purezza. Sulla base multi-bene di filtrazione per la clearance lisato batterico e purificazione del DNA, questo metodo crea un processo semplificato per la preparazione del plasmide. Piastre filtranti a base di silice contenente i media possono essere facilmente lavorate per filtrazione sotto vuoto o centrifughe per produrre notevoli quantità di DNA plasmidico. Analisi quantitative determinare il DNA plasmidico purificato è sempre di alta qualità con una media di OD 260/280 rapporti di 1.97. Nel complesso, i rendimenti plasmide offrire DNA più puro per applicazioni a valle, come la sequenza e la clonazione. Questo metodo semplificato di utilizzo Piastre Advance Filter AcroPrep consente l&#39;elaborazione manuale, semiautomatica o completamente automatica.

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

Questo protocollo è un&#39;alternativa conveniente per chiarimenti efficiente parallelo e purificazione del DNA plasmidico da<em&gt; E. coli</em&gt; Culture. Il processo di Advance AcroPrep inizia con una piastra di chiarificazione del lisato ottimizzato filtro seguita da purificazione su un legame ad alta capacità di legame del DNA piastra del filtro.

Purificazione semplificata del DNA plasmidi da culture procariotiche

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

19.9K Views.  Pall Life Sciences.  中文 

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 approccio G-quadruplex del DNA-affinità per purificazione di enzimaticamente attiva 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 ...

Ricerca

Biochimica

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.

Purificazione del DNA plasmidi da colonie batteriche Utilizzando il 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.

Una purificazione e l'analisi di attività In Vitro per un ppGpp (p) sintetasi da Clostridium difficile

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

Immunologia e infezioni

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.

High-throughput Purificazione di affinità-tagged proteine ​​ricombinanti

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.

Saggi Reporter massicciamente parallelo in cellule di mammifero in coltura

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.

Analisi della stabilità dei plasmidi con microfluidica a goccioline open-source

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

Bioingegneria

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 %.

Utilizzo dell'assemblaggio in vivo per la costruzione di plasmidi ad alta efficienza

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.

Plasmidi derivati ​​da DNA Strand Displacement Cancelli per reti di reazione implementazione chimici

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.

Purificazione genoma a livello di extrachromosomal circolare DNA dalle cellule eucariotiche

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: Assemblaggio chimico mediante inserimento del sito enzimatico di recupero e restrizione di plasmide

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

Purificazione semplificata del DNA plasmidi da culture procariotiche

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Esplora Altri Video

Capitoli in questo video

Un approccio G-quadruplex del DNA-affinità per purificazione di enzimaticamente attiva G4 Resolvase1

Purificazione del DNA plasmidi da colonie batteriche Utilizzando il kit Qiagen Miniprep

Una purificazione e l'analisi di attività In Vitro per un ppGpp (p) sintetasi da Clostridium difficile

High-throughput Purificazione di affinità-tagged proteine ricombinanti

Saggi Reporter massicciamente parallelo in cellule di mammifero in coltura

Analisi della stabilità dei plasmidi con microfluidica a goccioline open-source

Utilizzo dell'assemblaggio in vivo per la costruzione di plasmidi ad alta efficienza

Plasmidi derivati da DNA Strand Displacement Cancelli per reti di reazione implementazione chimici

Purificazione genoma a livello di extrachromosomal circolare DNA dalle cellule eucariotiche

CAPRRESI: Assemblaggio chimico mediante inserimento del sito enzimatico di recupero e restrizione di plasmide

Purificazione semplificata del DNA plasmidi da culture procariotiche

Trascrizione

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un approccio G-quadruplex del DNA-affinità per purificazione di enzimaticamente attiva G4 Resolvase1

Purificazione del DNA plasmidi da colonie batteriche Utilizzando il kit Qiagen Miniprep

Una purificazione e l'analisi di attività In Vitro per un ppGpp (p) sintetasi da Clostridium difficile

High-throughput Purificazione di affinità-tagged proteine ​​ricombinanti

Saggi Reporter massicciamente parallelo in cellule di mammifero in coltura

Analisi della stabilità dei plasmidi con microfluidica a goccioline open-source

Utilizzo dell'assemblaggio in vivo per la costruzione di plasmidi ad alta efficienza

Plasmidi derivati ​​da DNA Strand Displacement Cancelli per reti di reazione implementazione chimici

Purificazione genoma a livello di extrachromosomal circolare DNA dalle cellule eucariotiche

CAPRRESI: Assemblaggio chimico mediante inserimento del sito enzimatico di recupero e restrizione di plasmide

High-throughput Purificazione di affinità-tagged proteine ricombinanti

Plasmidi derivati da DNA Strand Displacement Cancelli per reti di reazione implementazione chimici