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.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>AcroPrep Advance Filter plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8175 (1 mL) or PN 8075 (350 &mu;l)</td>
<td>3 &mu;m glass fiber 0.2 &mu;m Supor&reg; membrane for clarification</td>
</tr>
<tr>
<td>AcroPrep Advance Filter Plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8132</td>
<td>for DNA Binding</td>
</tr>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 5017</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pressure pump</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 13157</td>
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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

19.8K Aufrufe.  Pall Life Sciences.  中文 

Serie: Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

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 ...

Forschung

Biologie

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformation von Plasmid-DNA in E. coli mit dem Hitzeschock-Methode

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Reinigung von Mitochondrien aus Hefezellen

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Reinigung von Vorläufern aus Skelettmuskeln

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

Genotypisierung von Pflanzen und Tieren Proben ohne vorherige DNA-Aufreinigung

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Substrat Generation für Endonukleasen der CRISPR / Cas-Systeme

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

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 ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplification, Next-Generation-Sequenzierung und genomischer DNA Mapping von Retrovirale Integration Seiten

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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 ...