1. Lysate Abstand <ol><li> Wachsen E. coli-Kulturen mit der gewünschten Plasmid-DNA in Deep Well Platten (1 mL Kultur / well) in Luria Broth mit geeigneten Antibiotika über Nacht bei 37 ° C unter Schütteln verwandelt. </li><li> Pellet E. coli in Kulturplatte bei 5.000 xg für 10 Minuten, dann dekantieren. </li><li> Resuspendieren jedes Pellet in 100 ul Resuspensionspuffer (50 mM Tris-HCl, pH 8,0, 10 mM EDTA, 100 ug / mL RNase A). </li><li> 100 l / well Lysispuffer (200 mM NaOH, 1% SDS) und schütteln mit Schüttler für 2 Minuten. </li><li> 100 l / well Neutralisierungspuffer (3,0 M Kaliumacetat, pH 5,5) und schütteln Sie für 2 Minuten. </li><li> Transfer-Zelllysat zur Klärung Platte Lysat. </li><li> Legen DNA-Bindung Platte auf der Oberseite von 350 ul Sammlung Platte aufnehmen und in unteren Vakuumkammer. Montieren Vakuumkammer (siehe Abbildung 1). </li><li> Legen Sie Lysat Klärung Platte oben auf Vakuumapparatur. </li><li> Bewerben Vakuum bei 10 in. Hg (0,34 bar) und sammeln Filtrat in DNA-Bindung Platte. Verwenden Sie keine Vakuum größer als 12 Zoll Hg. (0,41 bar). </li><li> Demontieren Vakuumkammer. Platz 2 mL Abfallsammlung Platte im unteren Teil des Gerät. </li></ol> 2. DNA-Bindung <ol><li> Bewegen Sie die DNA-Bindung Platte an der Spitze der Verteiler (siehe Abbildung 2). </li><li> Geben Sie 300 ul / well Binding Buffer (6 M Guanidin-HCl) und Pipette auf und ab zu mischen. </li><li> Bewerben Vakuum [~ 5 Zoll Hg (0,17 bar) für langsame Vakuum] und entsorgen Filtrat. DNA ist nun verpflichtet, Membran. </li><li> Waschen mit 400 ul / well Waschpuffer (80% Ethanol). </li><li> Bewerben Vakuum, dann verwerfen das Filtrat. Entsorgen Sie nicht erforderlich, wenn mit 2 mL Sammlung Platte oder Filterung direkt zu verschwenden. </li><li> Wiederholen Sie waschen einmal. </li><li> Blot unteren Filterplatte auf saugfähigen Tuch zu trocknen. </li><li> Bewerben Vakuum noch einmal für 5-10 Minuten, um das Entfernen von Restalkohol zu gewährleisten. </li><li> Blot unten nach Entfernung von Ethanol Tröpfchen zu gewährleisten. </li></ol> 3. DNA Elution <ol><li> Fügen Sie 70 ul / Vertiefung Elutionspuffer (10 mM Tris-HCl, pH 8,0, 1 mM EDTA). Werden höhere DNA-Konzentration gewünscht wird, kann ein Volumen von 50 ul / Vertiefung reduziert werden, aber der durchschnittliche Gesamtertrag niedriger sein wird. </li><li> Die Platte bei Raumtemperatur für 1 Minute. </li><li> Gereinigte DNA kann entweder durch Vakuum oder Zentrifugation eluiert werden. </li><li> Vakuum-Methode: Zeigen saubere Sammlung Platte (DNase, RNase frei) in Vakuumkammer. Platzieren Sie den Filter an der Oberseite Vakuumkammer, gelten Vakuum bei 15 in. Hg (0,5 bar) für 1 Minute, bis alle Elutionspuffer durch die DNA-Bindung Platte passiert hat. Sammeln gereinigte DNA (siehe Abbildung 3). </li><li> Zentrifugation Methode: Zeigen Reinigungsfilter Platte oben auf saubere Sammlung Platte und Zentrifuge bei 1.000 xg für 5 Minuten. </li></ol> 4. Quantitative und qualitative Analysen <ol><li> Messen OD 260/280, berechnen Konzentration der gereinigten DNA. </li><li> Führen Agarosegel-Analyse der gereinigten pDNA. </li></ol> 5. Repräsentative Ergebnisse E. coli-Lysat geklärt Verwendung traditioneller Zentrifugation und Pall AcroPrep Advance-Lysat Klärung Filterplatten zeigte spürbare Verringerung der Schmutz von Ausgangsmaterial. (Abbildung 4). Plasmid-DNA bei drei Konzentrationen wurde in TE-Puffer versetzt und durch die AcroPrep Advance-Lysat Klärung Filterplatte. Ein Vergleich von Plasmid-DNA-Konzentration, Pre-und Post-Filtration ~ zeigt 100% Wiederherstellung (Abbildung 5) bei allen drei Konzentrationen. Um die Gewinnung von DNA aus E. geklärt demonstrieren coli-Lysat, Plasmid-DNA wurde in Rohlysat versetzt und dann durch Filtration geklärt. Das gleiche Plasmid-DNA wurde Zelllysat nach der Filtration zugesetzt. Die Elektrophorese wurde auf Aliquots von jeder Probe (Abbildung 6) durchgeführt. Eine einzige, klare Bande von ähnlicher Intensität aus jeder Probe zeigt wenig oder gar keine DNA-Verlust bei der Filtration-basierte Klärung Schritt. Kulturen von E. coli mit pCAT Plasmid-DNA wurden von E. gereinigt coli-Lysaten mit Pall 350 &amp;mgr; l und 1 ml DNA-bindende Filterplatten, sowie zwei andere Marken von 350 ul DNA-Bindung-Platten. Alle Reinigungen wurden durchgeführt, wie in dem vom Hersteller empfohlenen Protokoll spezifiziert. Die höchste DNA-Konzentration und Gesamtertrag aus Pall 1 mL AcroPrep Advance-DNA-Aufreinigung Filterplatte (Abbildung 7 und Tabelle 2) zu sehen. Obwohl Wettbewerber W zeigt zudem eine hohe DNA-Konzentration (155 ng / mL), ist die totale DNA-Ausbeute der niedrigsten (4,7 pg / well) aufgrund der niedrigen Wiederherstellungs-Volume, ~ 30 ul. Wettbewerber M gab die niedrigste Konzentration von DNA bei 108 ng / ul mit einer Gesamtausbeute von 5,6 pg / well. Agarose-Gel-Analyse von gereinigtem pCAT DNA aus den drei verschiedenen DNA-bindenden Platten ist in Abbildung 8 dargestellt. Jede Sammelprobe Volumen wurde auf 65 ul eingestellt werden, um Unterschiede in der Wiederherstellungs-Volume vor der Elektrophorese zu korrigieren. Alle Proben zeigen, Plasmid-DNA supercoiled, aber DNA-Ausbeute aus jedem der Filterplatten variiert. Abbildung 9 zeigt die vorbereitete DNA ist alles in supercoiled Form und hat eine ähnliche Konzentration. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Montage von Vakuum-Manifold für Lysate Klarstellung. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Montage von Vakuum-Manifold für die DNA-Binding. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Montage von Vakuum-Manifold für Vakuum-Elution. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Filtration vereinfacht Probe Klärung. Dreifachen Proben von 300 ul Rohlysat durch Vakuumfiltration (350 ul AcroPrep Advance-Lysat Klärung Filterplatte) oder traditionellen Zentrifugation geklärt. OD 320 vor und nach der Klärung gemessen. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Vollständige Wiederherstellung von pDNA nach dem Durchgang durch AcroPrep Advance-Lysat Klärung Filterplatte. 300 ul TE-Puffer mit pCAT bei 25, 50 und 100 ug / ml versetzt. DNA-Konzentration und Erholung von OD 260 vor und nach der Filtration, N = 2 berechnet. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Abbildung 6" /> Abbildung 6. Rückgewinnung von pCAT DNA nach Klärung. Gleiche Mengen von pUC19 in Lysat vor oder nach Klärung versetzt, dann verdünnt 1 bis 10 in TE-Puffer. Loaded 2 ul / Spur auf 1,2% Agarosegel. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Abbildung 7" /> Abbildung 7. Höhere Plasmid DNA-Ausbeute pro Well mit Pall AcroPrep Advance-DNA-Bindung Filterplatte als Konkurrent Platten. PCAT Plasmid-DNA Ausbeute (OD 260) mit angegeben DNA-Aufreinigung Platten mit 1 mL (Pall und Wettbewerber M) oder 1,5 ml (Wettbewerber W)-Nacht-Kultur DH5a. Reinigung mit Platte vom Hersteller empfohlenen Protokoll. Fehlerbalken zeigen Standardfehler (n&gt; 6). <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Abbildung 8" /> Abbildung 8. Agarose-Gel-Analyse von DNA pCAT am angegebenen DNA-Bindung Platte gereinigt. 1,2% Agarose-Gelelektrophorese des gereinigten pDNA. Gepoolten Proben aus drei verschiedenen Reinigungen, Eluat auf 65 ul nach der Reinigung, 1:10 eingestellt. Loaded 2 ul pro Bahn. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Abbildung 9" /> Abbildung 9. Qualität und Gewinnung von reiner DNA ähnlich, wenn Zentrifuge oder Vakuum-Verfahren für die endgültige Eluat Sammlung Agarosegelelektrophorese des gereinigten pDNA verwendet werden. Die vom Hersteller empfohlene Protokoll für AcroPrep Advance-Filterplatten wurde pDNA Reinigung gefolgt. Die Elution wurde durch Zentrifugieren bei 1.000 xg für 5 Minuten oder Vakuum mit 15 Zoll Hg (0,5 bar) für 2 Minuten durchgeführt.

Die Autoren dieses Video-Artikel sind die Mitarbeiter von Pall Life Sciences, der das Instrument und Reagenzien in der Video-und Text-featured produzieren.

Wir beschreiben eine einfache, optimierte Protokoll AcroPrep Advance-Filterplatten, dass eine Reihe von Vorteilen bietet. Diese Methode bietet ein Maximum an Plasmid-DNA liefert, wenn im manuellen, teilautomatisierten oder vollautomatisierten AcroPrep Advance-Formate verarbeitet. Ein integrierter Vorfilter auf dem Lysat Abstand Platte sorgt für konsistente Filtration von Proben, auch solche mit einem hohen Gehalt an Partikeln. Die Well-Geometrie der Platte führt zu einer schnelleren, gleichmäßigeren Abscheidegrade über die Platte mit minimalem Totvolumen und die Steckdose Spitzengeometrie bietet direkten Durchfluss von Proben in den Receiver Platte ohne Bedenken von Kreuzkontamination. Wichtig ist, dass die hohe Bindekapazität Membran in DNA-Bindung Teller AcroPrep Advance-Filterplatten für maximale Rückgewinnung hochwertiger Plasmid-DNA optimiert.

<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

Wir beschreiben den gesamten Prozess der AcroPrep Advance-Filterplatten für 96 Plasmid-Präparationen, ab prokaryotischen Kultur und endend mit hoher Reinheit DNA. Basierend auf Multi-Well-Filtration für Bakterienlysat Luft-und DNA-Aufreinigung, erstellt diese Methode eine optimierten Prozess für Plasmid-Präparation. Filter-Platten mit Silica-basierte Medien können durch Vakuum-Filtration oder Zentrifuge nennenswerte Mengen von Plasmid-DNA-Ausbeute verarbeitet werden. Quantitative Analysen bestimmen die gereinigte Plasmid-DNA wird konsequent von hoher Qualität mit einer durchschnittlichen OD 260/280 Verhältnisse von 1,97. Insgesamt bieten Plasmid Erträge reiner DNA für nachgelagerte Anwendungen wie Sequenzierung und Klonierung. Dieser optimierte Methode zur Verwendung von AcroPrep Advance-Filterplatten ermöglicht die manuelle, halbautomatische oder vollautomatische Verarbeitung.

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

Dieses Protokoll ist eine kostengünstige Alternative für eine effiziente parallele Klärung und Plasmid-DNA Aufreinigung aus<em&gt; E. coli</em&gt; Kulturen. Die AcroPrep Advance-Prozess beginnt mit einer optimierten Lysat Klärung Filterplatte durch Reinigung auf eine hohe Bindekapazität DNA-Bindung Filterplatte gefolgt.

Optimierte Reinigung von Plasmid-DNA aus prokaryontischen Kulturen

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 Aufrufe.  Pall Life Sciences.  中文 

Serie: 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.

Ein G-Quadruplex-DNA-Affinität Ansatz zur Reinigung von enzymatisch aktiven 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 ...

Forschung

Biochemie

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.

Purifying Plasmid-DNA aus Bakterien-Kolonien mit dem 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.

Eine Reinigung und In vitro- Aktivität Assay für ein (p) PpGpp Synthestase von Clostridium difficile

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

Immunologie und Infektion

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 Reinigung von Affinity-markierter rekombinanter Proteine

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.

Entwicklung antiviraler Wirkstoffe mittels Oberflächenplasmonenresonanz

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

Biotechnik

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.

Plasmidstabilitätsanalyse mit Open-Source-Tröpfchen-Mikrofluidik

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 %.

Verwendung von In-vivo-Assemblierung für den hocheffizienten Plasmidbau

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.

Plasmid stammende DNA-Strand Displacement Gatter zum Implementieren 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 ...

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.

Genomweite Reinigung von extrachromosomale zirkuläre DNA aus eukaryotischen Zellen

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: Chimären Assembly durch Plasmid Recovery und Restriktion Enzym Site Insertion

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