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

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

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

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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

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

Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

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

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

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

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Vidéo: 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.

Purifiant l&#39;ADN plasmidique des colonies bactériennes en utilisant le kit Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

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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 d&#39;ADN plasmidique dans E. coli en utilisant la méthode du choc thermique

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.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

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.

Purification des mitochondries de cellules de levure

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.

Purification des progéniteurs du muscle squelettique

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.

Le génotypage des échantillons de plantes et d&#39;animaux sans purification d&#39;ADN Avant

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.

Génération substrat pour des endonucléases de CRISPR / Cas Systems

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.

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

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

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, Séquençage de nouvelle génération, et l&#39;ADN génomique Cartographie des rétrovirales Sites d&#39;intégration

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.

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

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Methods Article

Purification simplifiée d'ADN plasmidique de cultures procaryotes