1. Liquidación lisado <ol><li> Crecer E. culturas coli transformadas con el ADN de plásmido deseado en profundidad placas (1 ml de la cultura / pocillo) en caldo Luria con los antibióticos apropiados durante la noche a 37 ° C con agitación. </li><li> Pellet E. coli en la placa de cultivo a 5.000 xg durante 10 minutos, luego decantar el sobrenadante. </li><li> Resuspender cada pellet en 100 l de búfer Resuspensión (50 mM Tris-HCl, pH 8,0, 10 mM EDTA, 100 mg / ml RNasa A). </li><li> Añadir 100 tampón de lisis l / pocillo (200 mM NaOH, 1% SDS) y agitar con agitador de placas durante 2 minutos. </li><li> Añadir 100 l / pocillo de búfer neutralización (3,0 M de acetato de potasio, pH 5,5) y agitar durante 2 minutos. </li><li> Transferencia de lisado de células de la placa de lisado aclaración. </li><li> Coloque la placa de unión al ADN en la parte superior de la placa de recolección de 350 l, y el lugar en el fondo del colector de vacío. Montar colector de vacío (ver Figura 1). </li><li> Lisado lugar aclarar la placa en la parte superior de los aparatos de vacío. </li><li> Aplicar un vacío de 10 pulgadas de mercurio (0,34 bar) y recoger filtrado en la placa de unión al ADN. No utilice vacío superior a 12 mm Hg. (0,41 bar). </li><li> Desmontar colector de vacío. Coloque 2 ml de residuos recogida en la placa inferior del aparato. </li></ol> 2. De unión con ADN <ol><li> Mover la placa de unión del ADN a la parte superior de la placa (ver Figura 2). </li><li> Añadir 300 l Buffer / Encuadernación y (6 M guanidina-HCl) y una pipeta de arriba a abajo para mezclar. </li><li> Aplicar un vacío [~ 5 mm Hg (0,17 bar) para el vacío lento] y desechar filtrado. ADN está ligado a la membrana. </li><li> Lavar con 400 l / pocillo de tampón de lavado (80% de etanol). </li><li> Aplicar un vacío, a continuación, descartar el filtrado. Deseche innecesaria si se usa 2 colecta ml o filtrando directamente a la basura. </li><li> Repetir el lavado una vez. </li><li> Blot parte inferior de la placa de filtro sobre una toalla absorbente para secar. </li><li> Aplicar un vacío, una vez más durante 5-10 minutos para asegurar la eliminación de los restos de alcohol. </li><li> Blot fondo para asegurar la eliminación de las gotas de etanol. </li></ol> 3. Elución del ADN <ol><li> Añadir 70 l / y tampón de elución (10 mM Tris-HCl, pH 8,0, 1 mM EDTA). Si la mayor concentración de ADN que se desea, el volumen se reduce a 50 l / bien, pero el rendimiento total promedio será menor. </li><li> Incubar la placa a temperatura ambiente durante un minuto. </li><li> ADN purificado se eluye ya sea por vacío o centrifugación. </li><li> Método de vacío: Coloque la placa de recolección de limpia (DNasa, RNasa libre) en el colector de vacío. Colocar la placa de filtro en la parte superior del colector de vacío, aplicar vacío a 15 pulgadas de mercurio (0,5 bar) durante 1 minuto hasta que todos los buffer de elución ha pasado a través de la placa de unión al ADN. Recoger ADN purificado (ver Figura 3). </li><li> Método de centrifugación: Colocar la placa de purificación de filtro en la parte superior de la placa de recolección de limpia y se centrifuga a 1000 xg durante 5 minutos. </li></ol> 4. Los análisis cuantitativos y cualitativos <ol><li> Medida de OD 260/280, calcular la concentración de ADN purificado. </li><li> Realizar un análisis en gel de agarosa de la pDNA purificada. </li></ol> 5. Resultados representante E. coli lisado clarificado mediante centrifugación tradicional y Pall AcroPrep adelantado placas aclarar lisado filtro mostraron una reducción apreciable de los desechos de material de partida. (Figura 4). ADN plásmido en tres concentraciones, se añadieron en buffer TE y pasa a través de la placa de Avance AcroPrep aclarar lisado filtro. Una comparación de la concentración de ADN plásmido, antes y después de la filtración, muestra a 100% de recuperación (Figura 5) en las tres concentraciones. Para demostrar la recuperación de ADN de aclarar E. coli lisado ADN plásmido, se añadieron en lisado crudo y se clarificarán por filtración. El ADN del plásmido mismo se añadió a la filtración después de lisado celular. La electroforesis se realizó en alícuotas de cada muestra (Figura 6). Una banda única y clara de similar intensidad en cada ejemplo muestra poca o ninguna pérdida de ADN durante la fase de clarificación y filtración basados. Los cultivos de E. coli que contiene el ADN del plásmido pCAT fueron purificados de E. coli lisados ​​con 350 l de Pall y 1 ml de ADN filtro de placas de unión, así como otras dos marcas de 350 placas de unión al ADN l. Todas las purificaciones se realizaron según lo especificado en el protocolo recomendado por el fabricante. La mayor concentración de ADN y la producción total se ven desde un Pall ml adelantado AcroPrep ADN de purificación de placas de filtro (Figura 7 y Tabla 2). A pesar de la competencia W también muestra la concentración de ADN de alto (155 ng / l), el rendimiento total de ADN es la más baja (4,7 mg / así) debido a la recuperación del volumen bajo, aproximadamente 30 mL. Competidor M dio la menor concentración de ADN a 108 ng / l con un rendimiento total de 5,6 ug / pocillo. Agarose análisis en gel de ADN purificado pCAT de las tres placas de unión al ADN diferente se muestra en la Figura 8. Cada volumen de la muestra recogida se ajustó a 65 l para corregir las diferencias en el volumen de recuperación antes de la electroforesis. Todas las muestras de ADN de plásmido superenrollado mostrar, pero varía el rendimiento de ADN de cada una de las placas de filtro. La Figura 9 muestra que el ADN es preparado en forma de superenrollado y tiene una concentración similar. <img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Figura 1" /> Figura 1. Asamblea del colector de vacío para el Esclarecimiento lisado. <img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Figura 2" /> Figura 2. Asamblea del colector de vacío para la unión de ADN. <img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Figura 3" /> Figura 3. Asamblea del colector de vacío para la elución de vacío. <img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Figura 4" /> Figura 4. Filtración simplifica la aclaración de la muestra. Tres muestras de 300 l de lisado crudo aclaró por filtración al vacío (350 l Avance AcroPrep lisado placa aclaración filtro) o centrifugación tradicional. OD 320 antes y después de la aclaración. <img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Figura 5" /> Figura 5. La recuperación total de pDNA tras el paso por adelantado AcroPrep placa de filtro lisado aclaración. 300 L de buffer TE enriquecida con pCAT a 25, 50 y 100 ug / mL. Concentración de ADN y la recuperación calculado a partir de 260 OD antes y después de la filtración, N = 2. <img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Figura 6" /> Figura 6. La recuperación de ADN pCAT después de la clarificación. Igual cantidad de pUC19 añadidos en lisado antes o después de la aclaración, entonces se diluye 1 a 10 en buffer TE. Cargado 2 l / carril en el 1,2% en gel de agarosa. <img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Figura 7" /> Figura 7. Mayor rendimiento de ADN plasmídico por pocillo con ADN manto adelantado Acroprep placa de unión que las placas de filtro de la competencia. PCAT rendimiento de ADN plásmido (OD 260) se indica con placas de purificación de ADN con 1 ml (Pall M y la competencia) o 1,5 mL (Competidor W) durante la noche de la cultura DH5a. Purificación mediante el protocolo recomendado por el fabricante de la placa. Las barras de error indican el error estándar (n&gt; 6). <img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Figura 8" /> Figura 8. Agarosa análisis en gel de ADN purificado pCAT en la placa de unión al ADN indicó. Agarosa al 1,2% electroforesis en gel de la pDNA purificada. Muestras combinadas de las tres purificaciones por separado, eluido ajustado a 65 l después de la purificación, diluido 1:10. Cargado 2 l por carril. <img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Figura 9" /> Figura 9. La calidad y la recuperación de ADN puro similares cuando los métodos de centrifugación o vacío se utilizan para la final de la electroforesis en gel de agarosa colección eluido de la pDNA purificada. Protocolo recomendado por el fabricante para el Avance AcroPrep filtro de placas se ha seguido para la purificación pDNA. La elución se realizó por centrifugación a 1.000 xg durante 5 minutos o una aspiradora con 15 pulgadas de mercurio (0,5 bar) durante 2 minutos.

Los autores de este video-artículo son empleados de Pall Life Sciences que producen los instrumentos y reactivos aparecen en el video y el texto.

Se describe un protocolo fácil, ágil de las placas de filtro AcroPrep anticipada que ofrece varias ventajas. Este método proporciona un rendimiento máximo de ADN plásmido cuando se procesan en formatos de avance manual, semiautomático o totalmente automatizado-AcroPrep. Un pre-filtro integrado en la placa de autorización lisado proporciona una filtración de las muestras, incluso las que contengan altos de partículas. La geometría y de los resultados de la placa más rápida, las tasas de filtración más uniforme a través de la placa con un mínimo de volumen de retención, y la geometría de la punta de salida proporciona un flujo directo de muestras en el plato receptor sin problemas de contaminación cruzada. Es importante destacar que la membrana de alta capacidad de unión de la placa de unión al ADN de AcroPrep adelantado placas de filtro está optimizado para una máxima recuperación de la calidad del ADN plásmido de alto.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>
<td>&nbsp;</td>		</tbody>
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streamlined purification of plasmid dna from prokaryotic cultures

Se describe el proceso completo de placas adelantado AcroPrep de filtro para 96 ​​preparaciones de plásmidos, a partir de la cultura procariotas y terminando con el ADN de alta pureza. Sobre la base de múltiples pozos de filtración para el despacho de lisado bacteriano y purificación del ADN, este método crea un proceso simplificado para la preparación de plásmido. Placas de filtro que contiene los medios de comunicación a base de sílice pueden ser fácilmente transformados por filtración al vacío o centrifugación para producir cantidades apreciables de ADN del plásmido. Los análisis cuantitativos determinar el ADN de plásmido purificado es siempre de alta calidad con un promedio de OD 260/280 ratios de 1,97. En general, los rendimientos de plásmido ADN ofrecen más puro para aplicaciones posteriores, como la secuenciación y clonación. Este método simplificado de la utilización de placas AcroPrep Avance del filtro permite el procesamiento manual, semiautomático o completamente automático.

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 es una alternativa rentable para la clarificación eficiente en paralelo y purificación del ADN plasmídico a partir de<em&gt; E. coli</em&gt; Culturas. El proceso de avance AcroPrep comienza con una placa de lisado aclaración optimizado filtro seguido por la purificación en una alta capacidad de unión al ADN vinculante placa de filtro.

Purificación simplificada de ADN plásmido de las Culturas procariotas

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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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.

Purificación del ADN plásmido de las colonias de bacterias Utilizando el kit de 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 ...

Investigación

Biología

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.

Transformación de ADN plásmido en E. coli utilizando el 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.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

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.

Purificación de las mitocondrias de las células de levadura

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.

Purificación de los progenitores del 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.

Aislamiento y purificación de Kinesin desde Drosophila Los embriones

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.

El genotipado de muestras de plantas y animales, sin previa purificación del ADN

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.

Sustrato Generación para endonucleasas 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.

Plásmido derivado de ADN Strand Displacement Puertas de Redes de reacción de Ejecución Química

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.

La purificación de todo el genoma de ADN circular Extracromosómica a partir de células eucariotas

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