Gostaríamos de agradecer à Fundação Canadense para Inovação, a British Columbia Fundo de Desenvolvimento do Conhecimento e das Ciências e Pesquisa de Engenharia National Council (NSERC) do Canadá para apoiar estudos em andamento em regiões de baixo oxigênio das águas oceânicas costeiras e aberto. JJW foi apoiado por bolsas de NSERC e DAW foi apoiado por bolsas de NSERC, Killam ea fundação TULA Centro financiado pela Diversidade Microbiana e Evolution. SL foi apoiado pelo Centro de fundação TULA financiado pela Diversidade Microbiana e Evolution.

Nota: Todos os reagentes utilizados neste protocolo são extraídos das existências de laboratório em pequenos stocks de trabalho-isto é muito importante para evitar a contaminação cruzada de amostras de seu DNA e contaminação dos estoques de laboratório. Além disso, quando pipetagem, certifique-se de trocar a ponteira para cada adição para evitar a contaminação cruzada. Part One: Lise Celular e Digestão <ol><li> Comece este protocolo por descongelamento dos filtros Sterivex que contêm biomassa planctônica anteriormente concentrada no gelo. Para simplificar, aqui descrevemos o procedimento para o processamento de um filtro individual. Na prática, recomenda-se o processamento de 16 ou menos filtros de cada vez. </li><li> Uma vez que o filtro tem descongelado, começar a primeira de duas incubações: uma para lisar as células e remover RNA, e um para quebrar proteínas. Para a primeira incubação, adicionar 100 mL de lisozima (125 mg em 1000 TE mL) e 20 ml de RNase A (10 mcg / ml) para o filtro. Feche o filtro com Parafilm e deixá-lo para rodar no forno de hibridação a 37 ° C por 1 hora. </li><li> Para a incubação seguinte, adicionar 100 mL proteinase K e 100 l SDS 20% para o filtro. Reseal usando Parafilm e deixá-lo para rodar 1-2 horas a mais no forno de hibridização, desta vez a 55 ° C. </li><li> Usando uma seringa 5cc, transferir o lisado do filtro Sterivex em um tubo Falcon 15 ml. Lavar o filtro com um tampão de lise ml e adicionar o líquido para o enxágüe lisado em seu tubo Falcon utilizando a seringa 5cc. Agora que você tem lisadas e digerido suas células, você está pronto para extrair seu DNA. </li></ol> Parte II: Extração de DNA <ol><li> O próximo passo é usar o método fenol-clorofórmio para extrair o DNA de seu lisado. Adicionar um volume igual (cerca de 3 ml) de Fenol: Clorofórmio: álcool isoamílico (IAA) ao tubo lisado, certificando-se de trocar a ponteira para cada adição para evitar a contaminação cruzada. Vortex por 10 segundos para misturar bem. </li><li> Girar o tubo a 2500 g por 5 minutos, certificando-se a centrífuga é equilibrado. Durante a centrifugação, a mistura de fenol-clorofórmio-lisado deve separar em duas camadas: uma camada orgânica contendo o clorofórmio, fenol e proteínas a partir de sua amostra, no fundo, e uma camada aquosa, que consiste em seu DNA, água e outros moléculas mais hidrofílica, em cima. </li><li> Transferir a camada aquosa (contendo o DNA) em um tubo de 15 ml nova Falcon. Tenha cuidado para não tocar a interface com a ponta da pipeta (sempre deixar uma pequena quantidade da camada aquosa para trás). </li><li> Para este novo tubo, adicionar igual volume (cerca de 3 mL) de clorofórmio: IAA, certificando-se de trocar a ponteira para cada adição para evitar a contaminação cruzada. Vortex por 10 segundos. Este passo ajuda a remover qualquer fenol remanescentes de sua amostra de DNA. </li><li> Giram a 2.500 g por 5 minutos ou até que fase aquosa é clara. Transferência de fase aquosa para um novo tubo Falcon rotulados e adicionar 1 ml de TE em pH 8,0, tendo o cuidado para não tocar a interface com a ponta da pipeta (sempre deixar uma pequena quantidade da camada aquosa para trás). Esta camada aquosa contém o extrato de DNA. </li></ol> Parte III: Concentração de DNA e lavar <ol><li> O próximo passo é lavar e concentrar a amostra de DNA em um Amicon 15 ml tubo de centrifugação Ultra. Os tubos Amicon consiste de um filtro que retém as moléculas igual ou superior a um peso molecular especificados (o retentado) e um tubo para travar o fluxo-through (o filtrado). Neste caso, o filtro retém o seu DNA (o retentado). Transferir o seu extrato de DNA a partir do passo 2.5 para o compartimento do filtro de um tubo Amicon Ultra. </li><li> Giram a 3.500 g por 10 minutos. Verifique se há menos de 1 ml de líquido retido no filtro Amicon. Se permanece mais retentado, encher o filtro com TE e girar novamente. Certifique-se usar uma ponteira nova para cada adição TE para evitar a contaminação cruzada. </li><li> Remova o fluxo-through, ou filtrado, para outro tubo Falcon e salve-o na geladeira até que você tenha confirmado seu produto final, DNA em um gel (Parte IV). </li><li> Adicionar 2 ml de tampão TE para o filtro Amicon e spin em 3500 g por 6 minutos, certificando-se de usar uma ponteira nova para cada adição TE para evitar a contaminação cruzada. Piscina filtrado com que a partir do passo 3.3 e guardar no frigorífico. </li><li> Lavar o DNA mais duas vezes com o TE para um total de três lavagens (certificando-se de usar sempre uma ponta de pipeta fresco), e salvar o filtrado de cada vez (como passos por 3.3 e 3.4). Para a última lavagem, girar até 200 a 500 ml de retentado permanece no filtro Amicon. Registre o volume final e transferir o retentado (que contém o DNA) a um tubo de 1,5 ml rotulados Eppendorf. </li></ol> Parte IV: Determinação da Qualidade DNA <ol><li> Verificar a qualidade do DNA e estimar a concentração do DNA por esgotar-se as amostras em um gel 0,8% agarose gel staINED com 1 ml brometo de etídio (EtBr) durante a noite. (Seja extremamente cauteloso ao usar EtBr-it é um agente mutagénico conhecidos e possivelmente cancerígeno. Certifique-se de trocar as luvas com frequência e evitar EtBr transferência para o resto do laboratório e para a sua pele.) Carregar as amostras ao lado de escadas de DNA, que servem como tamanho e intensidade de normas (ver passo seguinte). </li><li> Nas pistas do primeiro de vários, carregar escadas com bandas de vários pesos moleculares e as concentrações. Recomendamos o padrão de carregamento seguinte: nas pistas do primeiro e último (pistas ultraperiféricas), mL de carga 10 de 50ng/μl de 1kb + ou escada 2log. Em faixas 2,3 e 4, mL de carga 2, 5 mL, e 10 ml, respectivamente, de 50ng/μl escada λHindIII. Por último, coloque 5 mL por via do extrato de DNA com corante para cada amostra. </li><li> Executar o gel a 15 volts por aproximadamente 16 horas (é recomendável executar durante a noite). </li><li> No dia seguinte, a fotografia do gel usando um sistema de documentação UV gel. Para determinar a faixa de peso molecular de seu DNA e sua concentração, compare as bandas de exemplo para as bandas das escadas. DNA de boa qualidade vai ter alto peso molecular (&gt; 36kb), e mostram pouca evidência de corte / degradação. Veja a Figura 1. </li></ol> Parte V: A centrifugação de gradiente de cloreto de césio <ol><li> Se a qualidade do DNA é bom ea quantidade é suficiente, você pode prosseguir para purificar o DNA através da realização de um cloreto de césio (CsCl) centrifugação de gradiente. Observe que esta etapa de purificação é opcional e não é necessário para todas as aplicações a jusante (por exemplo, se você deseja gerar bibliotecas fosmid você DEVE purificar, ao passo que se você simplesmente deseja executar reações de PCR, purificação geralmente não é necessário). Primeiro, nomeie um tubo de centrifugação para cada amostra. </li><li> A cada tubo de centrífuga, adicionar 160 mg de CsCl, 178 mL de DNA genômico. Após a adição de CsCl e DNA para o tubo, coloque um pequeno pedaço de parafilme em cima do tubo e inverter cuidadosamente o tubo de 1-20 vezes para misturar os componentes. Não misture por pipetagem, que pode causar ruptura do DNA. Em seguida, adicione 10 ml de 10 mg / mL EtBr a este tubo e misturá-lo da mesma maneira. (Seja extremamente cauteloso ao usar EtBr-it é um agente mutagénico conhecidos e possivelmente cancerígeno. Certifique-se de trocar as luvas com frequência e evitar EtBr transferência para o resto do laboratório e para a sua pele.) </li><li> Certifique-se que o peso de todos os tubos são equilibradas antes da centrifugação de tal forma que as diferenças de peso entre os tubos são menos de 1 mg. </li><li> Coloque os tubos no rotor utilizando hemostat, feche a tampa e coloque o rotor dentro da ultracentrífuga. </li><li> Feche a porta ultracentrífuga. Vácuo será aplicado assim que você fechar a porta. Executar durante a noite a 100 mil rpm por 18 horas e 20 ° C. </li><li> Centrifugação, quando estiver completa, retirar os tubos do rotor utilizando hemostat e coloque em uma cremalheira do tubo. </li><li> Visualizar o DNA com transiluminador de luz azul, ou se não estiver disponível, usar a luz UV de comprimento de onda longa, em um quarto escuro. Usar um par de óculos de âmbar filtro para ver a banda DNA. Veja a Figura 2. </li><li> Usando uma seringa e agulha estéril 1cc (26G 08/05), remova banda DNA e colocá-lo em um tubo Eppendorf de 1,5 ml. </li><li> Para ajudar a recuperar a maior parte do DNA preso no espaço morto da seringa, lavar a seringa com 100 mL de TE e adicionar a solução de lavado ao tubo. Prepare um tubo com 100 mL de buffer TE por amostra, a fim de evitar a contaminação cruzada. </li><li> Para remover EtBr do DNA, adicionar igual volume de água saturada de butanol ao tubo, inverter os tubos suavemente 1-20 vezes, centrifugar a 10000 rpm por 1 minuto e descartar a camada superior. </li><li> Repita a lavagem até a cor do butanol é transparente, 3-4 vezes mais. </li><li> Coloque 4 ml TE em um tubo de centrífuga Amicon Ultra e adicionar a solução de DNA a partir de cima. </li><li> Centrifugar a 3.500 g em temperatura ambiente, até que o volume de DNA é reduzida a cerca de 100-500 mL (aproximadamente 6-7 minutos) e descartar o escoamento. </li><li> Adicionar 2 ml de TE Amicon filtro e centrifugar a 3.500 g por 6 minutos, certificando-se de usar uma ponteira nova para cada adição para evitar a contaminação cruzada. Repita duas vezes. </li><li> Concentre-se para um volume final de 50-100 mL por centrifugação adicional quando necessário, e transferir a solução de DNA no filtro para um novo tubo de 1,5 ml Eppendorf. </li><li> Adicionar 40 mL de TE o filtro Amicon e pipetar cima e para baixo ao longo de ambas as membranas de filtro para eliminar qualquer DNA restantes. Adicionar esta solução para o tubo no 5.15. </li><li> Coloque um microcontrolador YM-30 unidade de filtro para um tubo de microcontrolador e pré-lavagem do microcontrolador, adicionando 200 mL de água autoclavada e centrifugação a 10000 rpm por 7 minutos. </li><li> Adicionar a solução de DNA a partir de 5,16 para o microcontrolador pré-lavados, a fim de concentrar ainda mais o DNA. Centrifugar a 5000-10000 g por 1-3 minutos. Verificar a quantidade de líquidona centrifugação filtro e repetir até que a quantidade de líquido no filtro é reduzida a aproximadamente 50 ul. </li><li> Coloque a unidade de filtro de cabeça para baixo em um tubo novo microcontrolador e centrifugar a 1000 g por 3 minutos. A quantidade ideal de solução concentrada é de 50-60 mL. </li><li> Medir e registrar a concentração do DNA em um Nanodrop e verificar a qualidade de pico (deve ser 260nm). </li><li> Transferir uma alíquota pequena do DNA para um tubo eppendorf limpo para trabalhar ações e congelar a -20. Transferir o restante do DNA a um segundo eppendorf limpo e congelar a menos 80. </li></ol> Resultados representativos: Quando este protocolo é feito corretamente, você deve ver uma imagem de gel após a etapa 4.4 no Determinação da qualidade do DNA semelhante à Figura 1. Concentração de DNA real de extratos irá variar dependendo da fonte da amostra. Após o passo 5,7 em CsCl centrifugação de gradiente, DNA genômico iluminado por uma luz azul deve ser semelhante à Figura 2. <img src="/files/ftp_upload/1352/1352Fig1newer.png" alt="Figura 1" /> Figura 1. 0,8% agarose imagem eletroforese em gel de DNA de alto peso molecular coletadas de quatro profundidades no subarctic Oceano Pacífico (em duplicado), manchado com o agente intercalando brometo de etídio (10 mg / ml). Gel foi executado em 15V para ~ 16hrs em tampão 1X TBE gel em execução. Bandas da amostra são de boa qualidade mostrando pouca evidência de corte mecânico (mostra faixas ou manchas único em oposição a várias bandas), embora os extratos 10m reter alguns RNA transitar (ver esfregaço na faixa Kb 0,5-2,0). <img src="/files/ftp_upload/1352/1352Fig2.jpg" alt="Figura 2" /> Figura 2. Band DNA genômico iluminado por uma luz azul após centrifugação em 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>

Dependendo do número de amostras devem ser processadas, isso pode ser um procedimento demorada devido aos dois passos de incubação de uma hora, e as lavagens repetidas e passos de centrifugação. É melhor plano de dois dias inteiros para este procedimento para deixar bastante tempo. Se houver problemas para obter o volume extrato final no tubo Amicon para reduzir até 200 500μl durante a concentração de DNA e de lavar roupa, tentar fazer lavagens adicionais TE e centrifugações (repetir Parte III) até que o vo...

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

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		<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 é usado para extrair o DNA genômico de alto peso molecular a partir da biomassa planctônica concentrou-se em 0,22 mM Sterivex filtros que foram tratados com armazenamento / tampão de lise e arquivados a -80 ° C, e para purificar esse DNA usando um gradiente de densidade de césio cloreto. O protocolo começa com duas etapas de uma hora de incubação para liberar o DNA das células e remover RNA. Em seguida, uma série de Fenol: Clorofórmio Clorofórmio e extrações são realizadas seguida de centrifugação para remover proteínas e componentes da membrana celular, a coleta do extrato aquoso de DNA, e as etapas de vários buffer de troca para lavar e concentrar o extrato. Parte V descreve a purificação opcional através de gradiente de cloreto de césio densidade. Recomenda-se a trabalhar com menos de 15 amostras de uma só vez para evitar confusão e reduzir o tempo de protocolo. O tempo total necessário para este protocolo depende do número de amostras a serem extraídas. Por 10-15 amostras e assumindo o equipamento de centrifugação adequada estiver disponível, este protocolo inteiro deve levar três dias. Certifique-se que os fornos de hibridação definir a temperatura no início do processo.

Watch this Scientific Journal Video about DNA Extraction from 0.22 ÃŽÂ¼M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation at JoVE.com

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

Nós descrevemos um método para extração de DNA genômico de alto peso molecular a partir da biomassa planctônica concentrou-se em 0,22 mM filtros Sterivex, seguido por centrifugação em gradiente de cloreto de césio densidade para purificação.

Extração de DNA de 0,22 mM Sterivex Filtros e Césio cloreto de centrifugação em gradiente de densidade

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

29.1K visualizações.  University of British Columbia - UBC. Nós descrevemos um método para extração de DNA genômico de alto peso molecular a partir da biomassa planctônica concentrou-se em 0,22 mM filtros Sterivex, seguido por centrifugação em gradiente de cloreto de césio densidade para purificação.

Vídeo: DNA Extraction from 0.22 μM 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.

Extração de DNA genômico de alto peso molecular de solos e sedimentos

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

Biologia

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.

Extração de DNA de material parafinado Embedded para análises genéticas e epigenéticas

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.

Extração de DNA de alto peso molecular de Esteiras Microbianas

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

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

Extração e Análise de Cortisol de cabelo humano e do macaco

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

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.

A técnica de infiltração-centrifugação para Extração de apoplásticas Fluid das folhas da planta Usando<em&gt; Phaseolus vulgaris</em&gt; Como exemplo

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.

Um método de extração de DNA híbrido para a Avaliação de comunidades bacterianas Qualitativa e Quantitativa de amostras de produção de aves de capoeira

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

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificação, sequenciamento de próxima geração, e Genomic DNA Mapeamento das retrovirais Integração Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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.

Isolamento de filtração de Ácidos Nucleicos: Um Método de extracção de DNA simples e rápido

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

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.

Preparação de Núcleos de tecido parafina-encaixados formalina-fixados para RNA e DNA Extraction

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

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.

Coleção de esperma de qualidade diferencial usando centrifugação gradiente de densidade

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 ...