Nos gustaría agradecer a la Fundación Canadiense para la Innovación, el British Columbia Fondo de Desarrollo de conocimientos y el Nacional de Ciencias e Ingeniería de Investigación (NSERC) de Canadá para apoyar los estudios en curso en las regiones de bajo nivel de oxígeno de las aguas oceánicas costeras y abiertas. JJW fue apoyado por becas de NSERC y DAW fue apoyado por becas de NSERC, Killam y la Fundación Centro TULA financiado por la diversidad microbiana y la evolución. SL fue apoyada por el Centro TULA financiado por la Fundación para la Diversidad microbiana y evolución.

Nota: Todos los reactivos utilizados en este protocolo que se distribuyan de las existencias de laboratorio que trabajan en pequeñas poblaciones-esto es muy importante para evitar la contaminación cruzada de muestras de ADN y la contaminación de las reservas de laboratorio. Además, al pipetear, asegúrese de cambiar las puntas de la pipeta para cada adición para evitar la contaminación cruzada. Primera parte: lisis celular y la digestión <ol><li> Comience este protocolo por el deshielo de los filtros que contienen Sterivex biomasa planctónica previamente concentrada en el hielo. Para simplificar, aquí se describe el procedimiento para la tramitación de un filtro individual. En la práctica, se recomienda el procesamiento de 16 o menos filtros a la vez. </li><li> Una vez que el filtro se ha derretido, comenzará el primero de dos incubaciones: una para lisar las células y eliminar el ARN, y uno para romper las proteínas. Para la primera incubación, añadir 100 l de lisozima (125 mg en 1000 l TE) y 20 ml RNasa A (10 mg / ml) en el filtro. Cerrar de nuevo el filtro con Parafilm y se deja de girar en el horno de hibridación a 37 ° C durante 1 hora. </li><li> Para la incubación siguiente, añadir 100 l de proteinasa K y 100 l 20% de SDS al filtro. Cerrar de nuevo con Parafilm y se deja de girar una o dos horas más en el horno de hibridación, esta vez a 55 ° C. </li><li> Con una jeringa de 5 cc, la transferencia del lisado del filtro Sterivex en un tubo Falcon de 15 ml. Enjuagar el filtro con una solución amortiguadora de lisis ml y agregar el líquido de enjuague para el lisado en su tubo Falcon con la jeringa de 5 cc. Ahora que ha lisan y digieren las células, usted está listo para extraer su ADN. </li></ol> Segunda parte: La extracción de ADN <ol><li> El siguiente paso es utilizar el método de fenol-cloroformo para extraer el ADN de su lisado. Añadir un volumen igual (alrededor de 3 ml) de fenol: cloroformo: alcohol isoamílico (IAA) en el tubo de lisado, asegurándose de que cambie la punta de la pipeta para cada Además de evitar la contaminación cruzada. Vortex durante 10 segundos para mezclar bien. </li><li> Girar el tubo a 2.500 g durante 5 minutos, asegurándose de que la centrífuga se equilibra. Durante la centrifugación, la mezcla de fenol-cloroformo-lisado se separan en dos capas: una capa orgánica que contiene el fenol cloroformo, y las proteínas de la muestra, en la parte inferior y una capa acuosa, que consiste en su ADN, el agua y otros moléculas más hidrófilas, en la parte superior. </li><li> La transferencia de la capa acuosa (que contiene el ADN) en un nuevo tubo de 15 ml Falcon. Tenga cuidado de no tocar la interfaz con la punta de la pipeta (siempre dejar una pequeña cantidad de la capa acuosa detrás). </li><li> Con este nuevo tubo, añadir un volumen equivalente (aprox. 3 ml) de cloroformo: IAA, asegurándose de que cambie la punta de la pipeta para cada Además de evitar la contaminación cruzada. Vortex durante 10 segundos. Este paso ayuda a eliminar cualquier resto de fenol a partir de su muestra de ADN. </li><li> Giran a 2500 g por 5 minutos o hasta que la capa acuosa es clara. Transferencia de fase acuosa en un nuevo tubo, llamado Falcon, y añadir 1 ml de TE pH 8.0, teniendo cuidado de no tocar la interfaz con la punta de la pipeta (siempre dejar una pequeña cantidad de la capa acuosa detrás). Esta capa acuosa contiene el extracto de ADN. </li></ol> Tercera parte: La concentración de ADN y de lavado <ol><li> El siguiente paso es lavar y concentrar la muestra de ADN en un tubo de 15 ml centrífuga Amicon Ultra. Los tubos Amicon consiste en un filtro que retiene las moléculas iguales o superiores a un peso determinado molecular (el concentrado) y un tubo para captar el flujo a través de (el filtrado). En este caso, el filtro retiene su ADN (el material retenido). Transfiere tus extraer el ADN de paso de 2,5 a el compartimiento del filtro de un tubo de Amicon Ultra. </li><li> Giran a 3500 g durante 10 minutos. Asegúrese de que hay menos de 1 ml de líquido retenido en el filtro Amicon. Si se mantiene más concentrado, vuelva a llenar el filtro con TE y girar de nuevo. Asegúrese de usar una punta de pipeta para cada TE Además de evitar la contaminación cruzada. </li><li> Retire el flujo a través, o filtrado, a otro tubo Falcon y guardarlo en la nevera hasta que haya confirmado su producto final de ADN en un gel (cuarta parte). </li><li> Añadir 2 ml de buffer TE con el filtro Amicon y giran a 3.500 g durante 6 minutos, asegurándose de usar una punta de pipeta para cada adición TE para evitar la contaminación cruzada. Piscina con filtrado que desde el paso 3.3 y guardar en la nevera. </li><li> Lavar el ADN dos veces más con TE para un total de tres lavados (asegurándose de utilizar siempre una punta de pipeta), y el ahorro del filtrado cada vez (como pasos por 3.3 y 3.4). Para el último lavado, centrifugado hasta 200 a 500 ml de los restos de material retenido en el filtro Amicon. Registre el volumen final y la transferencia del concentrado (que contiene el ADN) a una etiqueta del tubo Eppendorf de 1,5 ml. </li></ol> Cuarta parte: Determinación de la calidad del ADN <ol><li> Comprobar la calidad del ADN y estimar la concentración del ADN mediante la ejecución de sus muestras en un gel de 0,8% en gel de agarosa estacionesINED con 1 ml de bromuro de etidio (EtBr) durante la noche. (Tenga mucho cuidado cuando se utiliza bromuro de etidio, que es un mutágeno conocido y carcinógeno. Asegúrese de cambiar los guantes con frecuencia y evitar EtBr traslado al resto del laboratorio y para la piel.) Cargar las muestras de ADN junto a las escaleras, que sirven como tamaño y la intensidad de las normas (ver paso siguiente). </li><li> En el primero de varios carriles, la carga de las escaleras con bandas de diferentes pesos moleculares y concentraciones. Se recomienda el patrón de carga siguientes: en el primero y el último carril (carril externo), carga de 10 l de 50ng/μl de 1kb + o escalera 2log. En los carriles de 2,3 y 4, l carga de 2, l 5 y 10 l, respectivamente, de 50ng/μl escalera λHindIII. Por último, la carga de 5 l por vía de extracción de ADN con tinte para cada muestra. </li><li> Correr el gel a 15 voltios durante aproximadamente 16 horas (se recomienda ejecutar durante la noche). </li><li> Al día siguiente, la fotografía del gel usando un sistema de documentación de gel UV. Para determinar el rango de peso molecular de su ADN y su concentración, comparar las bandas de la muestra a las bandas de las escaleras. ADN de buena calidad se han elevado peso molecular (&gt; 36 kb), y muestran poca evidencia de corte / degradación. Véase la figura 1. </li></ol> Parte Cinco: La centrifugación gradiente de cloruro de cesio <ol><li> Si la calidad del ADN es bueno y la cantidad es suficiente, se puede proceder a purificar el ADN mediante la realización de un cloruro de cesio (CsCl) gradiente de centrifugación. Tenga en cuenta que esta etapa de purificación es opcional y no es necesario que todas las aplicaciones posteriores (por ejemplo, si desea generar bibliotecas fosmid se debe purificar, mientras que si simplemente desea llevar a cabo reacciones de PCR, la purificación no suele ser necesario). Primero, etiquete un tubo de centrífuga para cada muestra. </li><li> Para cada tubo de centrífuga, añadir 160 mg de CsCl, 178 l de ADN genómico. Después de la adición de CsCl y el ADN en el tubo, coloque un pequeño trozo de parafina en la parte superior del tubo y suavemente invierta el tubo de diez a veinte veces para mezclar los componentes. No mezclar con la pipeta, que puede causar una ruptura del ADN. A continuación, añadir 10 l de 10 mg / l EtBr a este tubo y mezclar en la misma forma. (Tenga mucho cuidado cuando se utiliza bromuro de etidio, que es un mutágeno conocido y carcinógeno. Asegúrese de cambiar los guantes con frecuencia y evitar EtBr traslado al resto del laboratorio y para la piel.) </li><li> Asegúrese de que el peso de todos los tubos están en equilibrio antes de la centrifugación de tal manera que las diferencias de peso entre los tubos son inferiores a 1 mg. </li><li> Colocar los tubos en la pinza del rotor utilizando, cierre la tapa y colocar el rotor dentro de la ultracentrífuga. </li><li> Cierre la puerta de ultracentrifugación. Vacío se aplicará tan pronto como se cierre la puerta. Realizadas por la noche a 100.000 rpm durante 18 horas y 20 ° C. </li><li> Cuando se haya completado la centrifugación, sacar los tubos del rotor con pinza hemostática y colocarla en un bastidor de tubo. </li><li> Visualizar el ADN con transiluminador de luz azul, o en su defecto, utilizar la luz UV de longitud de onda larga, en un cuarto oscuro. Use un par de gafas de filtro de color ámbar para ver la banda de ADN. Véase la figura 2. </li><li> Con una jeringa estéril de 1 cc y una aguja (26G 5 / 8), quite la banda de ADN y lo coloca en un tubo Eppendorf de 1,5 ml. </li><li> Para ayudar a recuperar la mayor parte del ADN atrapado en el espacio muerto en la jeringa, enjuague la jeringa con 100 l TE y la solución de enjuagar el tubo. Preparar un tubo con 100 l de buffer TE por muestra con el fin de evitar la contaminación cruzada. </li><li> Para eliminar EtBr a partir del ADN, añadir un volumen igual de agua saturada de butanol en el tubo, invertir los tubos suavemente diez a veinte veces, se centrifuga a 10000 rpm durante 1 minuto y descartar la capa superior. </li><li> Repetir el lavado hasta que el color de butanol es transparente, 3 - 4 veces más. </li><li> Colocar 4 ml TE en un tubo de centrífuga Amicon Ultra y la solución de ADN de más arriba. </li><li> Centrifugar a 3500 g, a temperatura ambiente, hasta que el volumen del ADN se reduce a aproximadamente 100 a 500 l (aproximadamente 6-7 minutos) y descartar el filtrado. </li><li> Añadir 2 ml de TE Amicon filtro y centrifugar a 3500 g durante 6 minutos, asegurándose de usar una punta de pipeta para cada uno además de evitar la contaminación cruzada. Repita dos veces. </li><li> Concentrado a un volumen final de 50 a 100 l por centrifugación adicionales si es necesario y la transferencia de la solución de ADN en el filtro a un nuevo tubo Eppendorf de 1,5 ml. </li><li> Añadir 40 ml TE al filtro Amicon y una pipeta de arriba a abajo a lo largo de las dos membranas de filtro para lavar cualquier resto de ADN. Añadir esta solución en el tubo de 5,15. </li><li> Coloque una Microcon YM-30 unidad de filtro en un tubo de Microcon prelavado y el microcontrolador mediante la adición de 200 l agua esterilizada y centrifugar a 10.000 rpm durante 7 minutos. </li><li> Añadir la solución de ADN de 5,16 a la Microcon prelavado con el fin de concentrar aún más el ADN. Centrifugar a 5000-10000 g por 1 - 3 minutos. Compruebe la cantidad de líquidoel sistema de centrifugación de filtro y repetir hasta que la cantidad de líquido en el filtro se reduce a aproximadamente 50 microlitros. </li><li> Coloque la unidad del filtro boca abajo en un tubo de Microcon nuevas y centrifugar a 1000 g por 3 minutos. La cantidad ideal de la solución concentrada es de 50-60 l. </li><li> Medir y registrar la concentración del ADN en un Nanodrop y control de calidad del pico (deberían ser 260 nm). </li><li> Transferencia de una pequeña alícuota del ADN a un tubo eppendorf limpio para trabajar acciones y congelar a menos 20. Transferir el resto del ADN a un segundo eppendorf limpio y se congelan a menos 80. </li></ol> Los resultados representativos: Cuando este protocolo se realiza correctamente, debería ver una imagen de gel después del paso de 4,4 en la determinación de la calidad del ADN similar a la Figura 1. Concentración de ADN real de los extractos puede variar dependiendo de la fuente de la muestra. Después del paso de 5,7 centrifugación en gradiente de CsCl, el ADN genómico iluminado por la luz azul debe ser similar a la Figura 2. <img src="/files/ftp_upload/1352/1352Fig1newer.png" alt="Figura 1" /> Figura 1. Agarosa al 0,8% la imagen de electroforesis en gel de ADN de alto peso molecular recogidos en cuatro profundidades en el océano Pacífico subártico (por duplicado), se tiñeron con el agente intercalante bromuro de etidio (10 mg / ml). Gel se ha ejecutado a 15 V para ~ 16 horas en el amortiguador 1X TBE gel en funcionamiento. Las bandas de la muestra son de buena calidad que muestran poca evidencia de corte mecánico (solo muestra las bandas o manchas en lugar de múltiples bandas), si bien los extractos de 10 millones de retener parte del ARN llevan más (ver mancha en el rango de 0,5 a 2,0 Kb). <img src="/files/ftp_upload/1352/1352Fig2.jpg" alt="Figura 2" /> Figura 2. Banda de ADN genómico iluminado por la luz azul después de centrifugación en gradiente de CsCl.

<ol>
	<li>Beja, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+BAC+or+not+to+BAC:+marine+ecogenomics.">To BAC or not to BAC: marine ecogenomics.</a> Curr Opin Biotechnol. 15 (3), 187-190 (2004).</li><li>Beja, O., Suzuki, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+analysis+of+bacterial+artificial+chromosome+libraries+from+a+marine+microbial+assemblage.">Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage.</a> Environ Microbiol. 2 (5), 516-529 (2000).</li><li>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=Community+genomics+among+stratified+microbial+assemblages+in+the+oceans+interior.">Community genomics among stratified microbial assemblages in the oceans interior.</a> Science. 311, 496-503 (2006).</li><li>Stein, J. L., Marsh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+uncultivated+prokaryotes:+isolation+and+analysis+of+a+40-kilobase-pair+genome+fragment+from+a+planktonic+marine+archaeon.">Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.</a> J Bacteriol. 178 (3), 591-599 (1996).</li><li>Somerville, C. C., Knight, I. T., Straube, W. L., Colwell, R. R. <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,+rapid+method+for+the+direct+isolation+of+nucleic+acids+from+aquatic+environments.">A simple, rapid method for the direct isolation of nucleic acids from aquatic environments.</a> Appl. Environ. Microbiol. 55 (3), 548-554 (1989).</li></ol>

Dependiendo de cuántas muestras se van a procesar, esto puede ser un procedimiento que consumen mucho tiempo debido a los dos pasos de la incubación de una hora, y los lavados repetidos y pasos de centrifugación. Es mejor plan de dos días para este procedimiento para dejar un montón de tiempo. Si hay problemas para conseguir el volumen de extracto final en el tubo de Amicon para reducir hasta 200-500μl durante la concentración de ADN y el lavado, trate de hacer más lavados de TE y centrifugaciones (repetir la te...

A correction was made to "<a href="/video/1352">DNA Extraction from 0.22 &#181;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation</a>". A typo in a volume's measurement was corrected in step 1.2.
The step was corrected from:
Once the filter has thawed, begin the first of two incubations: one to lyse the cells and remove RNA, and one to break down proteins. For the first incubation, add 100 &#956;l lysozyme (125 mg in 1000 &#956;l TE) and 20 ml RNase A (10 &#956;g/ml) to the filter. Reseal the filter with Parafilm and leave it to rotate in the hybridization oven at 37&#176;C for 1 hour.
to:
Once the filter has thawed, begin the first of two incubations: one to lyse the cells and remove RNA, and one to break down proteins. For the first incubation, add 100 &#956;l lysozyme (125 mg in 1000 &#956;l TE) and 20 &#956;l RNase A (10 &#956;g/ml) to the filter. Reseal the filter with Parafilm and leave it to rotate in the hybridization oven at 37&#176;C for 1 hour.

A correction was made to <a href="/video/1352">DNA Extraction from 0.22 &#181;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation</a>. A typo in a volume's measurement was corrected in step 1.2.

Erratum: DNA Extraction from 0.22 &#181;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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>1 cc syringe (26G5/8 needle)</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309597</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe (26G5/8 needle)</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309597</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml eppendorf tubes</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>0030 125.150</td>
<td>&nbsp;</td>
<tr>
<td>15ml tubes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>CLS430791</td>
<td>&nbsp;</td>
<tr>
<td>5cc syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309603</td>
<td>&nbsp;</td>
<tr>
<td>Amicon&reg; Ultra-4 Centrifugal Filter Devices</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC801096</td>
<td>10,000 Nominal molecular weight limit</td>
</tr>
<tr>
<td>Butanol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A383-1</td>
<td>Make it water saturated (butanol:water = 1:1)</td>
</tr>
<tr>
<td>Centrifuge rotor, JA5.3 </td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>368690</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge, Avanti&reg; J-E</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>369003</td>
<td>&nbsp;</td>
<tr>
<td>Cesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4036</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform:IAA (24:1)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>25666-500ML</td>
<td>&nbsp;</td>
<tr>
<td>Ethidium Bromide (EtBr)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E1510-10ml</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E5134</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization oven</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>13-247-10</td>
<td>37&deg;C & 55&deg;C</td>
</tr>
<tr>
<td>Lysis Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.75 M sucrose, 40 mM EDTA, 50 mM Tris (pH 8.3)</td>
</tr>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L6876-5G</td>
<td>125 mg in 1000 &mu;l TE</td>
</tr>
<tr>
<td>Microcon&reg; Ultracel YM-30 Centrifugal Filter Unit</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>42410</td>
<td>50 bp cut off</td>
</tr>
<tr>
<td>Parafilm</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-374-10</td>
<td>&nbsp;</td>
<tr>
<td>Phenol:Chloroform:IAA (25:24:1)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>77617-500ML</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>19133</td>
<td>&nbsp;</td>
<tr>
<td>RNase A</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP2539-100</td>
<td>10 &mu;g/ml</td>
</tr>
<tr>
<td>Safe Imager&trade; Blue light transilluminator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S37102</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Dodecyl Sulfate (SDS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4509</td>
<td>20% SDS</td>
</tr>
<tr>
<td>Sterivex filters</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SVG010RS</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0389</td>
<td>&nbsp;</td>
<tr>
<td>Table top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5415D</td>
<td>&nbsp;</td>
<tr>
<td>TE buffer, pH 8.0</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trizma&reg; base</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge rotor, TLA 100</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>343847</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge tube (7X20mm, PC)</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>343775</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge tube rack</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>348302</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge, Optima&reg; Max</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

dna extraction from 0.22 &mu;m sterivex filters and cesium chloride density gradient centrifugation

Este método se utiliza para extraer el ADN de alto peso molecular genómico a partir de biomasa planctónica se concentró en 0,22 M filtros Sterivex que han sido tratados con buffer de almacenamiento / lisis y archivados a -80 ° C, y para purificar el ADN utilizando un gradiente de densidad de cloruro de cesio. El protocolo se inicia con dos etapas de incubación de una hora para liberar el ADN de las células y eliminar el ARN. A continuación, una serie de fenol: cloroformo y cloroformo extracciones se llevan a cabo, seguido por centrifugación para separar las proteínas y los componentes de la membrana celular, la recolección del extracto acuoso de ADN, y varias medidas de cambio de amortiguación para el lavado y concentrar el extracto. Quinta Parte describe la purificación opcional a través de gradiente de densidad de cloruro de cesio. Se recomienda trabajar con menos de 15 muestras al mismo tiempo para evitar la confusión y reducir el tiempo de protocolo. El tiempo total requerido para este protocolo depende del número de muestras que se extraen. De 10 a 15 muestras y suponiendo que el equipo de centrifugación adecuada está disponible, este protocolo completo debe tomar 3 días. Asegúrese de tener los hornos de hibridación para configurar la temperatura en el inicio del proceso.

Watch this Scientific Journal Video about DNA Extraction from 0.22 Î¼M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation at JoVE.com

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

Se describe un método para la extracción de ADN de alto peso molecular genómico a partir de biomasa planctónica se concentró en 0,22 micras filtros Sterivex, seguido por centrifugación en gradiente de densidad de cloruro de cesio para la purificación.

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 ...

dna-extraction-from-022-m-sterivex-filters-cesium-chloride-density

Research

JoVE Journal

Biology

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

29.0K vistas.  University of British Columbia - UBC. Se describe un método para la extracción de ADN de alto peso molecular genómico a partir de biomasa planctónica se concentró en 0,22 micras filtros Sterivex, seguido por centrifugación en gradiente de densidad de cloruro de cesio para la purificación.

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

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 ...

Investigación

Biología

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 ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

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 ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

La técnica de infiltración-centrifugación para extracción de apoplástico fluido procedente de hojas de la planta Uso<em&gt; Phaseolus vulgaris</em&gt; Como ejemplo

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

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 ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

El aislamiento de filtración de ácidos nucleicos: A Extracción de ADN método sencillo y rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Recogida de esperma de calidad diferencial mediante centrifugación de gradiente de densidad

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

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 ...

Aislamiento mejorado de vesículas extracelulares bacterianas para microbiota multiómica

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Autor destacado: Aceleración de la investigación sobre la separación y heterogeneidad de vesículas extracelulares bacterianas 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.