Este trabajo fue financiado por el Programa Nacional Science Foundation Ambiental Genómica (Grant No. EF-0723707).

1. La extracción de células microbianas: <ol><li> Homogeneizar las alfombras microbianas con un mortero estéril molienda mezclando a fondo. Lugar aproximadamente a los 30 g (peso húmedo) de material de alfombra homogeneizada en un recipiente estéril de Waring batidora, añadir aproximadamente 100 ml de 1 M de NaCl (o una concentración específica de la muestra en uso), y la mezcla de tres veces a velocidad media durante 1 minuto con enfriamiento intermitente en un congelador a -20 ° C durante 1 min. La transferencia de la mezcla en una botella de 250 ml de centrífuga y llenar el volumen vacío restante con 1 M NaCl. Prepare una solución de NaCl en autoclave de agua DI y el filtro de esterilización de la misma. </li><li> Además desprender las células microbianas de la matriz por agitación (150 rpm) usando un vórtice (Vortex-Genie R 2) a temperatura ambiente durante 30 min. </li><li> Centrifugar a baja velocidad (500x g) durante 15 min a 4 ° C. Suavemente transferir el sobrenadante a un matraz con el menor trastorno posible a los sedimentos. </li><li> Utilizando el sedimento pellets, repita los pasos de fusión y 1.2 y 1.3 cuatro veces más con la adición de NaCl fresco a cada uno de los sedimentos de pellets en la final de la etapa 1.3. El sobrenadante de cada extracción de NaCl se transfiere a un frasco de dulce. </li><li> Combine los sobrenadantes de la extracción de células y centrifugar 5 en alícuotas de 200 ml a alta velocidad (25.000 xg) durante 15 min a 4 ° C. Descartar el sobrenadante, resuspender cada pellet de células en 10 ml de hexametafosfato de sodio al 2%, se combinan pastillas y completar el volumen a 200 ml con hexametafosfato de sodio al 2%. Lavar las células de agitación (150 rpm) a temperatura ambiente durante 30 min. Al final de este paso, debe tener un solo tubo de células para proceder al siguiente paso. </li><li> Se centrifuga a alta velocidad (25.000 xg) durante 15 min a 4 ° C. Descartar el sobrenadante, resuspender sedimento celular en 200 ml de TE (50 mM EDTA), y lavar las células de agitación (150 rpm) a temperatura ambiente durante 10 min. </li><li> Se centrifuga a alta velocidad (25.000 xg) durante 15 min a 4 ° C, descartar el sobrenadante, resuspender en 15 ml de TE (10 mM EDTA), y la tienda de 200 l alícuotas a -80 ° C hasta que sea necesario para la extracción de ADN. </li></ol> 2. Extracción de ADN y purificación: <ol><li> Añadir 200 l de NaCl 5 M, 200 l 10% de SDS, y 100 l 14,3 M β-mercaptoetanol a la alícuota microbiana de 200 l de células. Mezclar suavemente por inversión de 4.6 veces. </li><li> Someter la mezcla a 3 rondas de congelación-descongelación sumergiendo en nitrógeno líquido durante 2 min seguido de un descongelamiento en un baño de agua de 65 ° C durante 5 min. Ampliar última descongelación a 10 min. </li><li> Añadir 200 l de acetato de potasio 5 M (pH 5,5) y el lugar en hielo durante 10 min. Centrifugar a 10.000 xg durante 10 min a 4 ° C y la transferencia del sobrenadante a un nuevo tubo con una punta de la pipeta de gran calibre. Añadir 3 l de RNasa A y se incuba a 37 ° C durante 1 hora. </li><li> Añadir un volumen igual de cloroformo, agitar brevemente por inversión y centrifugar a 15.000 xg durante 10 min a temperatura ambiente. </li><li> Transferir cuidadosamente la capa superior del sobrenadante a un tubo nuevo con una punta de la pipeta de gran calibre. Evite el pipeteo de la capa de interfaz de color blanco situado entre las capas superior e inferior. Repita el procedimiento de extracción de cloroformo. </li><li> Añadir un volumen igual de frío (a -20 ° C) isopropanol al sobrenadante e incubar en hielo durante 30 minutos para precipitar el ADN. </li><li> Centrifugar a máxima velocidad (20.800 xg) durante 10 min a 4 ° C para precipitar el ADN. </li><li> Descartar el sobrenadante y lavar el ADN precipitado con 1 ml de frío (a -20 ° C) de etanol al 70%. </li><li> Centrifugar a máxima velocidad (20.800 xg) durante 10 min a 4 ° C, descartar el sobrenadante, repetir el 70% de etanol de lavado y secado al aire pellet de ADN por 10 min. Repita el 70% de etanol de lavado. </li><li> Resuspender el ADN en 25 l TE (10 mM EDTA), caliente a 65 ° C durante 5 minutos, se combinan en un tubo si varios tubos se han utilizado, y el maquillaje volumen a 500 ml con TE. </li><li> Añadir 500 l helado (o menos 4 ° C) 20% de glicol polietileno (PEG) (preparado en 1,2 M NaCl). Mezclar suavemente por inversión e incubar en hielo durante 10 min y centrifugar a máxima velocidad durante 10 minutos a 4 ° C para precipitar el ADN. Retire y deseche el sobrenadante, lavar el ADN granulado con 1 ml de etanol frío al 70%, de secado al aire durante 10 minutos, y resuspender en 30-50 l TE o agua de grado molecular. Facilitar la resuspensión de ADN por calentamiento a 65 º C durante 5 minutos y el ADN de la tienda a -80 ° C. </li></ol> 3. La pureza del ADN, la concentración y determinación del tamaño: <ol><li> Determinar la calidad del ADN mediante la medición del A y A 260/280 260/230 ratios usando un espectrofotómetro Nanodrop 1000 o cualquier instrumentación apropiada. </li><li> Determinar la concentración de ADN utilizando Quant-iT kit de ADN de doble cadena de ensayo de acuerdo a las instrucciones del fabricante. </li><li> Determinar el tamaño de ADN utilizando el campo de pulsos electroforesis en gel. Prepare un 1% en gel de agarosa con TBE 0.5X y utilizar los siguientes parámetros en el CHEF Mapper XA sistema, el tiempo de cambio inicial de 0,35 s, un tiempo de cambio de final de 7,67 s, y 120 ° ángulo incluido de un gradiente de 6,0 V / cm en una r linealamplificación de los factores. Mancha de gel con 1 x SYBR Green I durante 30 minutos. Carga entre 700-1000 ng de ADN total en gel. Ver fig. 3 de una forma condensada de todo el protocolo. </li></ol> Los resultados representativos: Celda de extracción: Secuencial de los extractos de células microbianas (sobrenadantes) han demostrado que la turbidez de los extractos disminuye a medida que aumenta el número de extracciones. Esto sugiere una reducción en el número de células después de cada extracción de células sucesivas. Es importante señalar aquí es que cada paso célula de extracción adicional proporciona la oportunidad para la introducción de contaminantes, el número de extracciones de células debe ser minimizado para que el final precipitado de células es representativa de la comunidad microbiana en general. Otras fuentes de contaminación se controla mediante la adopción de técnicas adecuadas de laboratorio estéril. Por ejemplo, las soluciones se prepararon en autoclave agua desionizada y esterilizada filtro. Los contenedores fueron esterilizados con alcohol, autoclave, y se trata a la luz ultravioleta y la radiación ultravioleta reticulante. Concentración de ADN y la determinación de la calidad: El protocolo produjo aproximadamente 2 g de ADN de alto peso molecular (35-50 kb) (Fig. 4) por gramo de muestra de tapete con un A 260/280 ratio de 1,6, y un A 260/230 ratio de 0,7 (Tabla 1). Aunque la relación A 260 / 230 pareció ser baja, no hay inhibición de aguas abajo de base molecular de aplicaciones como PCR-amplificación de los genes del 16S rRNA se observó en un estudio separado 7. Es importante tener en cuenta que el ADN de las esteras hipersalinas tiene dos fuentes principales de contaminación, EPS y las sales. Por tanto, es posible que los restos de estos contaminantes pueden estar influyendo en la A 260/230 relaciones a pesar de los enormes esfuerzos para reducir sus efectos sobre las aplicaciones de aguas abajo. La determinación del tamaño del ADN: Electroforesis en gel de campo de pulsos utiliza un flujo de corriente pulsante con interruptores intermitentes de dirección que resulta en una prueba de ADN, como se muestra en la figura 3. Nuestro protocolo produjo un ADN de alto peso molecular de aproximadamente 35 a 50 kb. Mientras que algunos de tamaño de ADN puede ser menor de 30 kb, es importante que una parte de la mancha por encima de 35 kb, ya que la clonación fosmid requiere ~ 40 kb inserciones de ADN y grandes fragmentos de ADN proporcionan un mayor acceso a las vías de biosíntesis intacto. En nuestros estudios, la prueba de ADN por encima de 35 kb se cortó y se purifica para la clonación de vectores de gran inserción y otras aplicaciones molecular. <img src="/files/ftp_upload/2887/2887fig1.jpg" alt="Figura 1"/> Figura 1. Microbianas hipersalinas alfombra sitio de muestreo (Laguna Grande) se encuentra en Eleuthera, Bahamas. <img src="/files/ftp_upload/2887/2887fig2.jpg" alt="Figura 2"/> Figura 2. Una sección de la alfombra microbiana hipersalina utilizados en este estudio. El tapete microbiano se obtuvo de una laguna hipersalina situada en Eleuthera, Bahamas. <img src="/files/ftp_upload/2887/2887fig3.jpg" alt="Figura 3"/> Figura 3. Representación esquemática de los procedimientos involucrados en la extracción de células microbianas, la lisis celular, la extracción y purificación del ADN metagenómica. <a href="http://www.jove.com/files/ftp_upload/2887/2887fig3large.jpg">Haga clic aquí para ver una imagen más grande.</a> <img src="/files/ftp_upload/2887/2887fig4.jpg" alt="Figura 4"/> Figura 4. Caracterización peso molecular del ADN extraído utilizando el campo de pulsos electroforesis en gel. Carril M es un marcador de alto peso molecular, y las calles 1 y 2 son repeticiones de ADN extraído de la metagenómica alfombra Eleuthera hipersalinos utilizando el protocolo anterior. <table border="1"><tbody><tr><td> Método de extracción </td><td></td><td> Concentración ng / g </td><td> A 260 / 280 </td><td> A 260 / 230 </td></tr><tr><td> PEG </td><td> Representante de una </td><td> 1806.1 </td><td> 1.64 </td><td> 0.76 </td></tr><tr><td></td><td> Rep 2 </td><td> 2010.8 </td><td> 1.61 </td><td> 0.75 </td></tr></tbody></table> Tabla 1. Medición de la concentración y la calidad del ADN extraído de alfombra microbiana hipersalinos.

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	<li>Decho, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilms+in+intertidal+systems:+an+overview.">Microbial biofilms in intertidal systems: an overview.</a> Cont. Shelf Res. 20, 1257-1273 (2000).</li><li>Dupraz, C., Visscher, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+lithification+in+marine+stromatolites+and+hypersaline+mats.">Microbial lithification in marine stromatolites and hypersaline mats.</a> Trends Microbiol. 13, 429-438 (2005).</li><li>Steffan, R. J., Goksoyr, J., Boj, A. K., Atlas, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+DNA+from+soils+and+sediments.">Recovery of DNA from soils and sediments.</a> Appl. Environ. Microbiol. 54, 2908-2915 (1988).</li><li>Lee, Y. K., Kim, H. W., Liu, C. L., Lee, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+DNA+extraction+from+marine+bacteria+that+produce+extracellular+materials.">A simple method for DNA extraction from marine bacteria that produce extracellular materials.</a> J. Microbiol. Methods. 52, 245-250 (2003).</li><li>Roose-Amsaleg, C. L., Garnier-Sillam, E., Harry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+Purification+of+Microbial+DNA+from+Soil+and+Sediment+Samples.">Extraction and Purification of Microbial DNA from Soil and Sediment Samples.</a> Appl. Soil Ecol. 18, 47-60 (2001).</li><li>de Lipthay, J. R., Enzinger, C., Johnsen, K., Aamand, J., S&#248;rensen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+DNA+extraction+method+on+bacterial+community+composition+measured+by+denaturing+gradient+gel+electrophoresis.">Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis.</a> Soil Biol. Biochem. 36, 1607-1614 (2004).</li><li>Bey, B. S., Fichot, E. B., Dayama, G., Decho, A. W., Norman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+High+Molecular+Weight+DNA+from+Microbial+Mats.">Extraction of High Molecular Weight DNA from Microbial Mats.</a> Biotechniques. 49, 631-640 (2010).</li><li>Kakirde, S. K., Parsley, L. C., Liles, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+does+matter:+Application-driven+approaches+for+soil+metagenomics.">Size does matter: Application-driven approaches for soil metagenomics.</a> Soil Biology &#38; Biochemistry. 42, 1911-1923 (2010).</li><li>Rodon, M. R., August, P. R., Bettermann, A. D., Brady, S. F., Grossman, T. H., Liles, M. R., Loiacono, K. A., Lynch, B. A., MacNeil, I. A., Minor, C., Tiong, C. L., Gilman, M., Osburne, M. S., Clardy, J., Handelsman, J., Goodman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+the+Soil+Metagenomic:+A+Strategy+for+Accessing+the+Genetic+and+Functional+Diversity+of+Uncultured+Microorganisms.">Cloning the Soil Metagenomic: A Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms.</a> Appl. Environ. Microbiol. 66, 2541-2547 (2000).</li><li>Beja, O., Aravind, L., Koonin, E. V., Suzuki, M. T., Hadd, A., Nguyen, L. P., Jovanovich, S. B., Gates, C. M., Feldman, R. A., Spudich, J. L., Spudich, E. N., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Rhodopsin:+Evidence+for+a+New+Type+of+Phototrophy+in+the+Sea.">Bacterial Rhodopsin: Evidence for a New Type of Phototrophy in the Sea.</a> Science. 289, 1902-1906 (2000).</li></ol>

Teniendo en cuenta que la eliminación total de las células de las muestras de alfombra complejos y muy variados microbiana no es práctico, la principal preocupación es qué tan bien las células extraídas representar a la comunidad microbiana alfombra en general. En un estudio previo, PCR-DGGE análisis de microorganismos genes del 16S rRNA mostró que la eliminación de cinco pasos de células utilizadas en este protocolo de extractos de células que son representativos de la comunidad microbiana alfombra en gener...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> β-mercaptoetanol </td><td> Sigma-Aldrich </td><td> M3148 </td><td></td></tr><tr><td> Polietilenglicol 8000 </td><td> Promega </td><td> V3011 </td><td> 20% en 1,2 M NaCl </td></tr><tr><td> Acetato de potasio </td><td> Fisher Scientific </td><td></td><td> Fisher Scientific </td></tr><tr><td> Quant-iT ensayo dsDNA kit </td><td> Invitrogen </td><td> Q33130 </td><td></td></tr><tr><td> RNasa </td><td> Epicentro </td><td> MRNA092 </td><td></td></tr><tr><td> Cloruro de sodio </td><td> BDH productos químicos </td><td> BDH8014 </td><td> Conc apropiado. </td></tr><tr><td> Dodecilsulfato de sodio </td><td> Fisher Scientific </td><td> 03-500-509 </td><td> 10% en agua </td></tr><tr><td> hexametafosfato de sodio </td><td> EMD Chemicals </td><td> SX0583-3 </td><td> 2% en agua </td></tr><tr><td> TBE </td><td> Fisher Scientific </td><td> BP1333-1 </td><td></td></tr><tr><td> CHEF Mapper XA Sistema </td><td> Bio-Rad Laboratories </td><td> 170-3670 </td><td></td></tr><tr><td> NanoDrop 1000 espectrofotómetro </td><td> Thermo Scientific </td><td> ND-1000 </td><td></td></tr><tr><td> Vórtice </td><td> Scientific Industries Inc. </td><td></td><td></td></tr><tr><td> Crosslinker ultravioleta </td><td> UVP </td><td></td><td></td></tr><tr><td> Mezclador Waring </td><td> Waring laboratorio </td><td> LB10S </td><td></td></tr></tbody></table>

extraction of high molecular weight dna from microbial mats

Análisis de éxito y preciso y la interpretación de los datos de metagenómica depende de la extracción eficiente de alta calidad, la comunidad de ADN de alto peso molecular (HMW). Sin embargo, las muestras ambientales colchoneta a menudo plantean dificultades para la obtención de grandes concentraciones de alta calidad, de alto peso molecular del ADN. Tapetes microbianos hipersalinos contienen altas cantidades de sustancias poliméricas extracelulares (EPS) 1 y las sales que pueden inhibir aplicaciones posteriores del ADN extraído. Los métodos directos y las duras a menudo se utilizan en la extracción de ADN de muestras de refractario. Estos métodos se utilizan normalmente debido a que la EPS en esteras, una matriz adhesiva, une el ADN 2,3 durante la lisis directa. Como resultado de los métodos de extracción más dura, el ADN se fragmenta en pequeños tamaños 4,5,6. El ADN se convierte en inadecuado para la reproducción de vectores de gran inserción. Con el fin de sortear estas limitaciones, se presenta una metodología mejorada para extraer el ADN de alto peso molecular de buena calidad y cantidad de tapetes microbianos hipersalinos. Se empleó un método indirecto, que supone la separación de las células microbianas de la matriz de alfombra de fondo a través de la mezcla y centrifugación diferencial. Una combinación de procedimientos mecánicos y químicos se utilizó para extraer y purificar el ADN de las células microbianas extraídos. El protocolo de los rendimientos de aproximadamente 2 g de ADN de alto peso molecular (35-50 kb) por gramo de muestra de alfombra, con un ratio de 1,6 260/280. Por otra parte, la amplificación de genes del 16S rRNA 7 sugiere que el protocolo es capaz de minimizar o eliminar los efectos inhibitorios de los contaminantes. Nuestros resultados proporcionan una metodología adecuada para la extracción de ADN de alto peso molecular de tapetes microbianos de los estudios de metagenómica funcional y puede ser aplicable a otras muestras ambientales de la extracción de ADN, que es un reto.

Watch this Scientific Journal Video about Extraction of High Molecular Weight DNA from Microbial Mats at JoVE.com

Extraction of High Molecular Weight DNA from Microbial Mats

Ofrecemos un protocolo mejorado para la extracción de ADN de alto peso molecular de tapetes microbianos hipersalinos. Las células microbianas son separados de la matriz de alfombra antes de la extracción y purificación del ADN. Esto mejora la concentración, calidad y tamaño de la DNA. El protocolo puede ser utilizado para otras muestras de refractario.

La extracción de ADN de alto peso molecular a partir de tapetes microbianos

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

extraction-of-high-molecular-weight-dna-from-microbial-mats

Research

JoVE Journal

Biology

19.4K vistas.  Arnold School of Public Health, University of South Carolina. Ofrecemos un protocolo mejorado para la extracción de ADN de alto peso molecular de tapetes microbianos hipersalinos. Las células microbianas son separados de la matriz de alfombra antes de la extracción y purificación del ADN. Esto mejora la concentración, calidad y tamaño de la DNA. El protocolo puede ser utilizado para otras muestras de refractario.

Video: Extraction of High Molecular Weight DNA from Microbial Mats

Biology of Microbial Communities  - Interview

Biología de las comunidades microbianas - Entrevista

Investigación

Biología

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

DNA Extraction from 0.22 &amp;mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

Extracción de ADN de 0,22 M Filtros Sterivex y centrifugación de densidad de cloruro de cesio Gradient

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

La extracción de ADN de alta molecular genómico de peso de los suelos y los sedimentos

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Extracción de ADN a partir de material incluido en parafina para análisis genéticos y epigenéticos

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Un híbrido método de extracción de ADN para la Evaluación de las comunidades bacterianas cualitativa y cuantitativa de Aves de Corral en muestras de producción

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

La mucina en gel de agarosa de electroforesis: Western Blotting para glicoproteínas de alta peso molecular


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Preparación de núcleos de tejido incluidas en parafina fijadas con formalina para ambos RNA y DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

Los métodos modificados para la carga de placas de extracción de ADN de alto rendimiento reducen el potencial de contaminación

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Aislamiento de miocitos aurículas y ventriculares murinas de alta calidad para mediciones simultáneas de transitorios de Ca2+ y corriente de calcio tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Extracción a pequeña escala de ADN genómico de Caenorhabditis elegans

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...