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

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

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

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
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<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>
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<td>AcroPrep Advance Filter Plate</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 8132</td>
<td>for DNA Binding</td>
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<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>PALL</td>
<td>PN 5017</td>
<td>&nbsp;</td>
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<td>Vacuum pressure pump</td>
<td>&nbsp;</td>
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streamlined purification of plasmid dna from prokaryotic cultures

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

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

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

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

Purificazione semplificata del DNA plasmidi da culture procariotiche

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

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JoVE Journal

Biology

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Video: 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.

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

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

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Biologia

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.

Trasformazione del DNA plasmidi in E. coli con il metodo Heat Shock

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.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

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.

Purificazione dei mitocondri da cellule di lievito

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.

Purificazione di progenitori dal muscolo scheletrico

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

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolamento e purificazione di chinesina dal Drosophila Embrioni

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

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.

Genotipizzazione di campioni di piante e degli animali, senza purificazione del DNA Prima

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.

Generazione Substrato per endonucleasi di CRISPR / Cas Sistemi

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.

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

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

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Purificazione genoma a livello di extrachromosomal circolare DNA dalle cellule eucariotiche

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