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 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

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 ...

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Research

JoVE Journal

Biology

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

29.0K 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 &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.

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 ...

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 ...

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 ...

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 ...

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.

Extração em pequena escala de Caenorhabditis elegans DNA genômico

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

Isolamento Aprimorado de Vesículas Extracelulares Bacterianas para Microbiota Multi-Ômica

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 em destaque: acelerando a pesquisa sobre separação e heterogeneidade 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.