우리는 해안과 열린 바다 바닷물의 낮은 산소 지역에 지속적인 연구를 지원하기위한 혁신, 브리티시 컬럼비아 기술 개발 기금과 국립 과학 및 캐나다의 공학 연구 협의회 (NSERC)에 대한 캐나다 재단 감사드립니다. JJW가 NSERC와 도스의 장학금이 지원되었고 NSERC, 킬람와 툴라 기초 미생물 다양성과 진화에 대한 재정 지원 센터에서 장학금으로 지원했다. SL은 미생물 다양성과 발전을위한 기초 자금 툴라 센터에 의해 지원되었다.

참고 :이 프로토콜에서 사용되는 모든 시약이 작동 주식을이 당신의 DNA 샘플 및 연구소 주식 오염의 교차 오염을 방지하는 것이 매우 중요 작은 실험실로 주식에서 aliquoted 있습니다. 또한, pipetting 때 교차 오염을 피하기 위해 모든 외에도 위해 피펫 팁을 변경할 수 있는지 확인하십시오. 부 원 : 세포 용해와 소화 <ol><li> 얼음 이전에 집중 planktonic의 바이오 매스를 포함 Sterivex 필터를 해동하여이 절차를 시작합니다. 단순 보려면 여기를 우리는 개별 필터를 처리를 위해 절차를 설명합니다. 실제로, 우리는 한 번에 처리 16 또는 이하의 필터를 권장합니다. </li><li> 필터가 해동되면 두 incubations의 첫 시작 : 하나는 세포를 lyse하고 단백질을 분해하는 RNA, 그리고 하나를 제거합니다. 첫 부화 들어, 필터 100 μl 라이 소 자임 (1000 μl TE 125 MG) 20 ML RNase (10 μg / ML)을 추가합니다. Parafilm와 필터를 봉인하고 37 하이브 리다이 제이션 오븐에 ° C 1 시간을 회전 둡니다. </li><li> 다음 부화 들어, 필터 100 μl Proteinase의 K 100 μl 20% SDS를 추가합니다. 55 봉인 그것이 하이브 리다이 제이션 오븐에 1-2시간 더 회전 Parafilm를 사용 떠나, 이번 ° C. </li><li> 5cc 주사기를 사용하여 15 ML 팔콘 튜브로 Sterivex 필터의 lysate를 전송합니다. 1 ML의 용해 버퍼와 필터를 씻어가 5cc 주사기를 사용하여 팔콘 튜브의 lysate에 액체 린스 추가할 수 있습니다. 이제 lysed하고 세포를 소화했는지, 당신은 그들의 DNA를 추출 준비가되어 있습니다. </li></ol> 두 번째 부분 : DNA 추출 <ol><li> 다음 단계는 당신의 lysate에서 DNA를 추출하기 위해 페놀 - 클로로포름 방법을 사용하는 것입니다. 교차 오염을 피하기 위해 각각의 이외에 대한 피펫 팁을 변경해야하고, lysate 튜브에 Isoamyl의 알코올 (IAA) : 클로로포름 : 페놀의 동일한 음량을 (3ml 정도) 추가합니다. 잘 섞어 10 초 와동. </li><li> 원심 분리기가 균형되어 있는지 확인하고, 5 분 2천5백g에서 튜브를 스핀. 아래에 샘플에서 페놀, 클로로포름 및 단백질을 포함하는 유기 층, 그리고 당신의 DNA, 물 구성되어 수성 층을, 및 기타 : 원심 분리하는 동안, 페놀 - 클로로포름 - lysate 혼합물은 두 레이어로 분리해야합니다 맨에 대한 자세한 친수성 ​​분자. </li><li> 새로운 15 ML 팔콘 튜브에 수성 레이어를 (당신의 DNA를 포함)의 전송. 피펫 팁 (항상 뒤에 수성 층의 작은 금액을 떠나)와 인터페이스를 만지지 않도록주의하십시오. </li><li> 교차 오염을 피하기 위해 각각의 이외에 대한 피펫 팁을 변경해야하고, IAA :이 새로운 튜브하려면, 클로로포름의 동일한 볼륨 (약 3mL)를 추가합니다. 10 초 와동. 이 단계는 당신의 DNA 샘플에서 남아있는 페놀을 제거하는 데 도움이됩니다. </li><li> 수성 층 분명히 때까지 5 분 또는 2천5백그램에서 스핀. 새로운 표시 팰컨 튜브에 수성 레이어를 전송하고 피펫 팁 (항상 뒤에 수성 층의 작은 금액을 떠나)와 인터페이스를 터치하지 않도록주의하고, pH를 8.0에서 TE 1 ML를 추가합니다. 이것은 수성 층은 DNA 추출물이 포함되어 있습니다. </li></ol> 부 세 : DNA 농도 및 세척 <ol><li> 다음 단계는 15 ML Amicon 울트라 원심 분리기 튜브의 DNA 샘플을 세척하고 집중하는 것입니다. Amicon 튜브가 (여과액)를 통해 흐름을 잡으려고 특정 분자량 (retentate)과 튜브에 이상 분자를 유지하는 필터로 구성되어 있습니다. 이 경우 필터는 DNA (retentate)을 유지합니다. Amicon 울트라 튜브 필터 구획 단계 2.5에서 DNA 추출물을 전송합니다. </li><li> 10 분 3,500g에 스핀. Amicon 필터에 보관 액체 미만의 1 ML이 있는지 확인하십시오. 더 retentate 남아 리필 TE와 필터와 다시 스핀 경우. 교차 오염을 피하기 위해 모든 TE 추가를위한 신선한 피펫 팁을 사용해야합니다. </li><li> 다른 팰컨 튜브로 흐름을 통해, 또는 여과수를 제거하고 젤에서 최종 DNA 제품을 확인까지 (파트 포) 냉장고에 저장합니다. </li><li> Amicon 필터 2 ML TE 버퍼를 추가하고 교차 오염을 피하기 위해 각각의 TE 추가를위한 신선한 피펫 팁을 사용해야하고, 6 분 3천5백g에 스핀. 풀 스텝 3.3 그걸로 여과물하고 냉장고에 저장합니다. </li><li> 세 세척 (항상 신선한 피펫 팁을 사용해야하는)의 총 TE와 함께 DNA 번 더 씻어하고, (사람마다 단계 3.3 및 3.4 등) 여과물 각 시간을 절약. 마지막 세척 들어, 스핀 Amicon 필터에 retentate 유해 200-500 ML까지. 최종 볼륨을 기록하고 표시 1.5 ML Eppendorf 튜브에 (당신의 DNA를 포함) retentate를 전송합니다. </li></ol> 부 네 : DNA 품질의 결정 <ol><li> DNA의 품질을 확인하고 젤 0.8 % 아가로 오스 겔 역에서 샘플을 실행하여 DNA의 농도를 추정하룻밤 1ml ethidium 브로마이드 (EtBr)로 ined. (사용하는 경우 매우주의 EtBr를 이것은 알려진 mutagen하고 의심 발암 물질입니다. 종종 장갑을 변경하고 실험실의 나머지 부분과 피부에 EtBr 전송을 방지해야합니다.)으로서 DNA 사다리 옆에있는 샘플을로드 크기와 강도 기준 (다음 단계 참조). </li><li> 처음 몇 차선에서, 다양한 분자 무게와 농도의 밴드와 함께 사다리를로드합니다. 처음이자 마지막 차선에서 (바깥쪽 차선), 1킬로바이트의 50ng/μl 부하 10 μl + 또는 2log 사다리 : 우리는 다음의 로딩 패턴을 권장합니다. 2,3 및 4 차선, 부하 2 μl, 5 μl 및 50ng/μl λHindIII의 사다리 각각 10 μl,에. 마지막으로, 각 샘플에 대한 염료와 DNA 추출물의 차선 당 5 μl를로드합니다. </li><li> 약 16시간 15 볼트 (우리가 야간 실행하는 것이 좋습니다)에서 겔을 실행합니다. </li><li> 그 다음날, UV 젤 문서 시스템을 사용하여 겔을 사진에 담아보세요. 당신의 DNA와 농도의 분자량 범위를 확인하려면 사다리의 밴드에 대한 샘플 밴드를 비교할 수 있습니다. 양질의 DNA 높은 분자량을 (&gt; 36킬로바이트) 있고, 전단 / 저하의 작은 증거를 보여줍니다. 그림 1을 참조하십시오. </li></ol> 부품 다섯 : 세슘 클로라이드 그라데이션 원심 분리 <ol><li> DNA 품질이 좋고 수량이 충분있다면, 당신은 세슘 염화물 (CsCl) 기울기 원심 분리를 수행하여 DNA를 정화 진행할 수 있습니다. 이 정화 단계는 선택 사항이며 모든 다운 스트림 응용 프로그램 (예를 들어, 당신이 단순히 PCR 반응을 수행하려는 경우 반면에, 정화는 보통 필요 없습니다, 당신이 정화서는 fosmid 라이브러리를 생성하려는 경우)에 필요한되지 않습니다. 첫째, 각 시료에 대해 하나 원심 분리기 튜브 레이블. </li><li> 각각의 원심 분리기 튜브하려면, CsCl, 게놈 DNA의 178 μl 160 MG를 추가합니다. 튜브에 CsCl와 DNA를 추가한 후, 튜브 위에 parafilm의 작은 조각을 배치하고 부드럽게 구성 요소를 혼합하기 위해 튜브에게 10-20 번 반전. pipetting으로 혼합하지 마십시오, 이는 DNA의 전단 원인이 될 수 있습니다. 다음,이 튜브 10 μg / μl EtBr 10 μl를 추가하고 같은 방식으로 그것을 섞는다. (사용하는 경우 매우주의 EtBr - 그것이 알려진 mutagen하고 의심 발암 물질입니다. 종종 장갑을 변경하고 실험실의 나머지 부분과 피부에 EtBr 전송을 방지해야합니다.) </li><li> 모든 튜브의 무게는 튜브 사이에 체중 차이가 1 MG보다하는 이러한 원심 분리하기 전에 균형 있는지 확인합니다. </li><li> 회전자를 사용 hemostat에 튜브를 삽입, 뚜껑을 닫고 초원 심 분리기 내부의 회전자를 놓습니다. </li><li> 초원 심 분리기 도어를 닫습니다. 진공, 당신은 빨리 문 닫아으로 적용됩니다. 18 시간 동안 100,000 RPM 20 ° C.에서 하룻밤 실행 </li><li> 원심 분리가 완료되면, 튜브 랙에 hemostat과 장소를 사용하여 회전자에서 튜브를 꺼내. </li><li> 푸른 빛의 transilluminator로 DNA를 시각화하거나 사용할 경우 어두운 방에서 긴 파장 UV 라이트를 사용합니다. DNA 밴드를 볼 수 황색 필터 안경을 착용하십시오. 그림 2를 참조하십시오. </li><li> 살균 1cc 주사기와 바늘을 (26G 8분의 5)를 사용하여 DNA 밴드를 제거하고 1.5ml Eppendorf 튜브에 놓으십시오. </li><li> 주사기의 죽은 공간에 갇혀있는 DNA의 대부분을 복구하기 위해, 100 μl TE와 주사기를 씻어 튜브에 씻어서 솔루션을 추가합니다. 교차 오염을 방지하기 위해 시료 100 μl TE 버퍼 하나의 튜브를 준비합니다. </li><li> DNA의 EtBr을 제거하려면 부드럽게 튜브 10-20 시간, 1 분 10,000 rpm으로 원심 분리기 반전, 튜브에 물이 포화 butanol의 동일한 볼륨을 추가하고 상위 레이어를 폐기하십시오. </li><li> 4 번 이상 - butanol의 색상은 3 투명 때까지 세척 반복합니다. </li><li> 플레이스 4 ML Amicon 울트라 원심 분리기 튜브의 TE하고 위에서 DNA 솔루션을 추가합니다. </li><li> DNA의 볼륨이 약 100-500 μl (약 6~7분)로 감소 때까지 상온에서 3,500g에서 원심과 흐름을 통해 폐기하십시오. </li><li> Amicon 필터 2ml TE를 추가하고 교차 오염을 피하기 위해 각 이외에 신선한 피펫 팁을 사용해야하고, 6 분 3,500그램에서 원심 분리기. 두 번 반복합니다. </li><li> 필요에 따라 추가 원심 분리에 의해 50-100 μl의 최종 볼륨에 집중하고 새로운 1.5 ML Eppendorf 튜브에 필터의 DNA 솔루션을 전송할 수 있습니다. </li><li> Amicon 필터 40 μl TE를 추가하고 나머지 DNA를 씻어 두 필터 세포막을 따라 위아래로 피펫. 5.15에있는 튜브에이 솔루션을 추가합니다. </li><li> 200 μl autoclaved 물을 추가 7 분 동안 10,000 rpm으로 centrifuging하여 Microcon 튜브 및 prewash Microcon에 Microcon YM - 30 필터 장치를 배치합니다. </li><li> 추가로 DNA를 집중 5.16에서 위해 prewashed Microcon에 DNA 솔루션을 추가합니다. 삼분 - 1 5천~1만g에서 원심 분리기. 액체의 양을 확인필터 및 반복 원심 분리에 필터에 액체의 양은 약 50 μl로 감소 때까지. </li><li> 새로운 Microcon 튜브에 거꾸로 필터 장치를 삽입하고 3 분 1,000그램에서 원심 분리기. 집중 솔루션의 이상적인 크기는 50-60 μl입니다. </li><li> 측정 및 Nanodrop에 DNA의 농도를 기록하고 (260nm한다) 최고의 품질을 확인합니다. </li><li> 마이너스 20 주식과 동결 일을위한 깨끗한 eppendorf 튜브에 DNA의 작은 나누어지는을 전송합니다. 마이너스 80에서 두 번째 깨끗한 eppendorf 및 동결에 DNA의 나머지 부분을 전송합니다. </li></ol> 대표 결과 : 이 프로토콜이 올바르게 완료되면, 당신은 1 그림과 유사한 DNA 품질의 결정 단계에서 4.4 후 젤 이미지를 볼 수 있습니다. 추출물의 실제 DNA 농도는 샘플의 소스에 따라 달라집니다. CsCl 기울기 원심 분리 단계에서 5.7 후, 파란 빛으로 조명 게놈 DNA 2 그림과 유사해야합니다. <img src="/files/ftp_upload/1352/1352Fig1newer.png" alt="그림 1" /> 그림 1. intercalating 에이전트 ethidium 브로마이드 (10 MG / ML) 물들일 아북극의 태평양 (중복)를, 네 개의 깊이에서 수집한 높은 분자량의 DNA의 0.8 % 아가로 오스 겔 전기 영동 이미지. 젤은 1X TBE 젤 실행 버퍼에 ~ 16hrs을위한 15V에서 실행되었다. 샘플 밴드는 기계적 전단 (싱글 밴드 또는 여러 밴드 반대로 얼룩을 보여줍니다)의 작은 증거를 보여주는 좋은 품질의 10m 추출물 일부 RNA가 (0.5-2.0 KB 범위에 얼룩 참조) 이월 유지하지만. <img src="/files/ftp_upload/1352/1352Fig2.jpg" alt="그림 2" /> 그림 2. CsCl 기울기 원심 분리 후 파란 광선에 의해 조명 게놈 DNA 밴드.

<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>

처리하는 방법에 많은 샘플 아르에 따라이 두 한 시간 동안 부화 단계 및 반복 세척과 원심 분리 단계로 인해 시간이 많이 소요되는 절차하실 수 있습니다. 충분한 시간을두고이 절차에 대한 이틀 동안 계획하는 것이 좋습니다. 해당 볼륨이 남은까지 (파트 3 번 반복) 추가 TE의 세척 및 centrifugations하고 시도, DNA 농도 및 세척하는 동안 200 500μl로 줄이기 위해 Amicon 튜브의 마지막 추출 볼륨을 받고 ?...

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

이 방법은 스토리지 / 용해 버퍼를 취급​​하고 -80 ° C에 보관되었습니다 0.22 μm의 Sterivex 필터에 집중 planktonic의 바이오 매스에서 높은 분자량의 게놈 DNA를 추출하고, 세슘 염화물 밀도 기울기를 사용하여이 DNA를 정화하는 데 사용됩니다. 프로토콜은 세포에서 DNA를 해방하고 RNA를 제거하는 방법은 두 한 시간 배양 단계를 시작합니다. 다음으로, 페놀 일련의 : 클로로포름 및 클로로포름 extractions는 단백질과 세포막의 구성 요소, 수성 DNA 추출물의 수집과 추출을 씻어하고 집중 여러 버퍼 교환 단계를 제거하기 위해 원심 분리 다음에 수행됩니다. 부품 다섯 세슘 염화물 농도 기울기를 통해 옵션 정화에 대해 설명합니다. 그것은 혼동을 피하기 및 프로토콜 시간을 줄이려고 한 번에 미만 15 샘플에서 작동하도록 권장합니다. 이 프로토콜에 필요한 총 시간은 추출되는 샘플 수에 따라 달라집니다. 10-15 샘플 및 적절한 원심 분리 장비를 사용할 수있는 가정의 경우이 모든 프로토콜 3 일이 소요됩니다. 당신은 프로세스의 초기에 온도로 설정 하이브 리다이 제이션 오븐을 가지고 있는지 확인하십시오.

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

우리는 정화를 위해 세슘 염화물 밀도 기울기 원심 분리 다음 0.22 μm의 Sterivex 필터에 집중 planktonic의 바이오 매스에서 높은 분자량의 게놈의 DNA의 추출을위한 방법을 설명합니다.

0.22 μm의 Sterivex 필터 및 세슘 클로라이드 밀​​도 기울기 원심 분리 장치에서 DNA 추출

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

29.0K 조회수.  University of British Columbia - UBC. 우리는 정화를 위해 세슘 염화물 밀도 기울기 원심 분리 다음 0.22 μm의 Sterivex 필터에 집중 planktonic의 바이오 매스에서 높은 분자량의 게놈의 DNA의 추출을위한 방법을 설명합니다.

비디오: 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.

소일스 및 세디먼츠에서 높은 분자량의 게놈의 DNA의 추출

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

연구

생물학

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.

유전과 Epigenetic 분석을위한 파라핀 임베디드 재료에서 DNA 추출

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.

미생물 매트에서 높은 분자량의 DNA의 추출

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.

추출 및 인간과 원숭이의 머리에서 코르티솔 분석

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.

공장에서 유체 Apoplastic의 추출을위한 침투-원심 분리 기술을 사용하여 잎<em&gt; 위 Phaseolus 심상</em&gt; 예를 들어

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.

가금류 생산 샘플에서 세균 공동체의 정성 및 정량 평가를위한 하이브리드 DNA 추출 방법

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.

레트로 바이러스 통합 사이트의 증폭, 차세대 시퀀싱 및 게놈 DNA 매핑

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.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

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.

RNA 및 DNA 추출 모두 포르말린 고정 파라핀 - 임베디드 조직 코어의 제조

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.

조밀도 기온 변화도 원심 분리를 사용 하 여 차동 품질의 정자 컬렉션

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 ...

0.22 μm의 Sterivex 필터 및 세슘 클로라이드 밀​​도 기울기 원심 분리 장치에서 DNA 추출

0.22 μm의 Sterivex 필터 및 세슘 클로라이드 밀도 기울기 원심 분리 장치에서 DNA 추출