1. Apuramento lisado <ol><li> Crescer E. culturas coli transformada com plasmídeo de DNA desejado no bem profundo placas (1 mL da cultura / poço) em caldo de Luria com antibiótico adequado durante a noite a 37 ° C com agitação. </li><li> E. coli Pellet em placa de cultura em 5.000 xg por 10 minutos, em seguida, o sobrenadante decantar. </li><li> Ressuspender cada pellet em 100 mL de buffer ressuspensão (50 mM Tris-HCl, pH 8,0, 10 mM EDTA, 100 mcg / mL de RNase A). </li><li> Adicionar 100 mL Tampão de Lise / poço (200 mM NaOH, 1% SDS) e agitar com placa shaker por 2 minutos. </li><li> Adicionar 100 mL de buffer Neutralização / poço (3,0 M acetato de potássio, pH 5.5) e agitar por 2 minutos. </li><li> Transferência lisado celular para lisado placa esclarecimento. </li><li> Colocar a placa de ligação ao DNA em cima de 350 mL prato de coleta e coloque em baixo do colector de vácuo. Montar coletor de vácuo (ver Figura 1). </li><li> Coloque placa de esclarecimento lisado em cima do aparelho de vácuo. </li><li> Aplicar vácuo a 10 pol Hg (0,34 bar) e coletar DNA filtrado para placa de ligação. Não use vácuo superior a 12 pol Hg. (0,41 bar). </li><li> Desmonte coletor de pó. Coloque 2 mL placa de coleta de lixo no fundo do aparelho. </li></ol> 2. DNA Binding <ol><li> Mover a placa de DNA de ligação ao topo do colector (ver Figura 2). </li><li> Adicionar 300 mL de buffer / poço Binding (6 M de guanidina-HCl) e uma pipeta cima e para baixo para misturar. </li><li> Aplicar vácuo [~ 5 pol Hg (0,17 bar) para vácuo lento] e descartar filtrado. DNA é agora obrigado a membrana. </li><li> Lavar com 400 mL / poço de tampão de lavagem (80% de etanol). </li><li> Aplicar vácuo, em seguida, descartar o filtrado. Descarte desnecessário se usar duas placas coleção mL ou filtragem diretamente para o lixo. </li><li> Repita lave uma vez. </li><li> Blot fundo da placa de filtro de papel absorvente para secar. </li><li> Aplicar vácuo mais uma vez por 5-10 minutos para assegurar a remoção de resíduo de álcool. </li><li> Blot fundo para assegurar a remoção de gotículas de etanol. </li></ol> 3. Eluição DNA <ol><li> Adicionar 70 mL tampão de eluição / poço (10 mM Tris-HCl, pH 8,0, 1 mM EDTA). Se maior concentração de DNA é desejado, o volume pode ser reduzido a 50 mL / poço, mas o rendimento médio total será menor. </li><li> Incubar a placa em temperatura ambiente por um minuto. </li><li> DNA purificado pode ser eluída por qualquer vácuo ou centrifugação. </li><li> Método de vácuo: Coloque placa da coleção clean (DNase, RNase free) no colector de vácuo. Colocar a placa de filtro em cima do colector de vácuo, aplique vácuo de 15 pol Hg (0,5 bar) por 1 minuto até que todos tampão de eluição passou através da placa de DNA de ligação. Coletar DNA purificado (ver Figura 3). </li><li> Método de centrifugação: Coloque placa de purificação do filtro em cima da placa da coleção clean e centrifugar a 1000 xg por 5 minutos. </li></ol> 4. As análises quantitativas e qualitativas <ol><li> Medida OD 260/280, calcular concentração de DNA purificado. </li><li> Realizar análise de gel de agarose do PDNA purificada. </li></ol> 5. Resultados representante E. coli lisado clarificado utilizando centrifugação tradicionais e Pall AcroPrep Adiantamento Placas lisado esclarecimento de filtro mostrou redução significativa de detritos a partir de matéria-prima. (Figura 4). DNA do plasmídeo em três concentrações foi cravado em tampão TE e passado através da placa de esclarecimento AcroPrep Adiantamento lisado filtro. Uma comparação da concentração de DNA plasmidial, pré e pós-filtração, shows ~ recuperação de 100% (Figura 5) em todas as três concentrações. Para demonstrar a recuperação do DNA de E. esclarecidas coli DNA, lisado plasmídeo foi cravado em lisado bruto e, em seguida, esclareceu por filtração. O mesmo DNA do plasmídeo foi adicionado à célula de filtração após lisado. Eletroforese foi realizada em alíquotas de cada amostra (Figura 6). A banda, único e claro de intensidade semelhante de cada amostra demonstra pouco caso de qualquer perda de DNA durante a etapa de clarificação filtração-based. Culturas de E. coli contendo DNA plasmídeo PCAT foram purificados a partir de E. coli lisados ​​usando mL da Pall 350 mL e 1 DNA de ligação placas de filtro, bem como duas outras marcas de 350 placas de DNA mL de ligação. Todas as purificações foram realizados conforme especificado no protocolo recomendado pelo fabricante. A maior concentração de DNA e rendimento total são vistos a partir mL da Pall AcroPrep um avanço placa DNA de purificação do filtro (Figura 7 e Tabela 2). Embora Concorrente W também mostra alta concentração de DNA (155 ng / mL), rendimento de DNA total é a mais baixa (4,7 mg / poço), devido ao volume de recuperação baixo, ~ 30 mL. Concorrente M deu a menor concentração de DNA de 108 ng / mL com um rendimento total de 5,6 mg / poço. Agarose gel análise de DNA purificado PCAT das três placas de DNA diferentes ligação é mostrado na Figura 8. Cada volume da amostra coletada foi ajustado para 65 mL para corrigir diferenças no volume de recuperação antes da eletroforese. Todas as amostras de DNA mostram superenrolado plasmídeo, mas o rendimento do DNA de cada uma das placas de filtro varia. Figura 9 mostra o DNA é tudo preparado na forma superenrolado e tem concentração similar. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Figura 1" /> Figura 1. Montagem de Manifold vácuo de Esclarecimento Lysate. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Figura 2" /> Figura 2. Montagem de Manifold de vácuo para Binding DNA. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Figura 3" /> Figura 3. Montagem de Manifold de vácuo para eluição de vácuo. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Figura 4" /> Figura 4. Filtração simplifica esclarecimento amostra. Triplicatas de 300 mL de lisado bruto clarificado por filtração a vácuo (350 mL AcroPrep Adiantamento placa esclarecimento lisado filtro) ou centrifugação tradicional. OD 320 medido antes e após esclarecimento. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Figura 5" /> Figura 5. Recuperação total dos PDNA após a passagem por Antecipação AcroPrep placa de filtro lisado esclarecimento. 300 mL tampão TE enriquecida com PCAT a 25, 50 e 100 mcg / mL. Concentração de DNA e recuperação calculados a partir de 260 OD antes e após a filtração, N = 2. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Figura 6" /> Figura 6. Recuperação de DNA PCAT após esclarecimento. Quantidades iguais de pUC19 cravado em lisado antes ou depois do esclarecimento, então diluída 1 a 10 em tampão TE. Loaded 2 mL / lane em 1,2% agarose gel. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Figura 7" /> Figura 7. Maior rendimento de DNA plasmidial por poço com pall DNA Avanço Acroprep placa de filtro de ligação de chapas concorrentes. Rendimento DNA PCAT plasmídeo (OD 260) usando placas de purificação de DNA indicou com 1 mL (Pall e M Concorrente) ou 1,5 mL (Concorrente W) cultura overnight de DH5α. Purificação usando o protocolo recomendado pelo fabricante chapa. Barras de erro indicam o erro padrão (n&gt; 6). <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Figura 8" /> Figura 8. Agarose gel análise de DNA purificado PCAT na placa de DNA indicaram vinculativo. 1,2% eletroforese em gel de agarose do PDNA purificada. Amostras combinadas de três purificações separado, eluato ajustada para 65 mL após purificação, 01:10 diluída. Loaded 2 mL por pista. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Figura 9" /> Figura 9. Qualidade e de recuperação do DNA puro que quando os métodos de centrifugação ou a vácuo são usados ​​para a final eluato eletroforese em gel de agarose coleção do PDNA purificada. Protocolo recomendado pelo fabricante de placas Adiantamento AcroPrep filtro foi seguido para a purificação PDNA. Eluição foi realizada por centrifugação a 1.000 xg por 5 minutos ou a vácuo com 15 pol Hg (0,5 bar) por 2 minutos.

Os autores deste artigo de vídeo-são funcionários da Pall Life Sciences que produzem os instrumentos e reagentes apresentado no vídeo e texto.

Nós descrevemos um protocolo, facile simplificada de placas de filtro AcroPrep antemão que oferece várias vantagens. Este método oferece rendimentos máximos DNA plasmídeo quando processados ​​em formatos Avanço manual, semi-automática ou totalmente automatizadas AcroPrep. Um pré-filtro integrada na placa de depuração lisado fornece filtração consistentes de amostras, inclusive aqueles contendo altos níveis de partículas. A geometria dos poços da placa de resultados no mais rápido, taxas de filtração mais uniforme em toda a placa com o volume mínimo de hold-up, ea geometria da ponta de saída fornece fluxo direto de amostras em placa de receptor sem preocupações de contaminação cruzada. Importante, a membrana de alta capacidade de ligação em chapa de DNA de ligação de placas Adiantamento AcroPrep filtro é otimizado para recuperação máxima de DNA de alta qualidade plasmídeo.

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		<div>
		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<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>
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<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>
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<td>Vacuum pressure pump</td>
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streamlined purification of plasmid dna from prokaryotic cultures

Descrevemos o processo completo de Placas Adiantamento AcroPrep Filtro para 96 ​​preparações plasmídeo, a partir de cultura procarióticas e terminando com DNA de alta pureza. Baseado em multi-filtração para o apuramento bem lisado bacteriano e purificação de DNA, esse método cria um processo simplificado para a preparação de plasmídeo. Placas de filtro à base de sílica contendo mídia pode facilmente ser processado por filtração a vácuo ou centrifugação para produzir quantidades apreciáveis ​​de DNA plasmidial. Análises quantitativas determinar o DNA purificado plasmídeo é consistentemente de alta qualidade com média de OD 260/280 índices de 1,97. No geral, os rendimentos plasmídeo oferecer DNA mais puro para aplicações a jusante, tais como seqüenciamento e clonagem. Este método simplificado de usar placas de Adiantamento AcroPrep filtro permite o processamento manual, semi-automática ou totalmente automatizadas.

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

Este protocolo é uma alternativa rentável para o esclarecimento paralela eficiente e purificação de DNA plasmidial de<em&gt; E. coli</em&gt; Culturas. O processo de Adiantamento AcroPrep começa com uma placa de filtro otimizado lisado esclarecimento seguida de purificação em uma ligação de alta capacidade de ligação DNA placa do filtro.

Purificação simplificada do DNA plasmidial das culturas Procarióticas

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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Vídeo: 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.

Purificação de DNA plasmidial colônias bacterianas Usando o Kit Qiagen Miniprep

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

Biologia

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.

Transformação do DNA plasmidial em E. coli Usando o método de choque térmico

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.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

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.

Purificação de mitocôndrias de células de levedura

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.

Purificação de Progenitores do músculo esquelético

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 Purificação de cinesina partir Drosophila Os embriões

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.

Genotipagem de amostras de plantas e animais, sem purificação DNA Antes

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.

Geração de substrato para Endonucleases de CRISPR / Cas Sistemas

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.

Strand DNA Deslocamento Portas para redes de reação de Execução químicos derivados do plasmídeo

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.

A purificação de todo o genoma de DNA circular Extracromossômica de células eucarióticas

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