이 작품은 국립 과학 재단 (National Science Foundation) 환경 게놈 프로그램 (그랜트 번호 EF - 0723707)에 의해 재정 지원되었다.

1. 미생물 세포 추출 : <ol><li> 철저하게 혼합하여 멸균 분쇄 유봉과 미생물 매트를 균질. 약 워링 믹서기의 멸균 용기에 무균 매트 재료의 모든 30g (서부 유럽 표준시 무게), 1 분 1 M NaCl (또는 사용 예제 특정 농도) 100 ML에 대한 추가하고, 중간 속도로 세 번 조화를 배치 1 분 -20 ° C의 냉동고에 간헐적으로 냉각. 250 ML의 원심 분리기 병에 슬러리를 전송하고 1 M NaCl과 함께 남아있는 빈 볼륨을 작성하십시오. autoclaved DI 워터의 NaCl 용액을 준비하고 소독 필터. </li><li> 또한 30 분 실온에서 vortexer (소용돌이 - 지니 R 2)를 사용하여 (150 RPM) 흔들림으로 모체의 미생물 세포를 이동시키다. </li><li> 4 15 분 ° C. 낮은 속도 (500x g)에서 원심 분리기 부드럽게 퇴적물에 최소한의 방해와 플라스크에 뜨는를 전송합니다. </li><li> pelleted 퇴적물을 사용하면, 단계 1.3의 끝 부분에 각각의 퇴적물 펠렛 신선한 NaCl의 추가와 함께 1.2 1.3 4 개의 추가 시간을 혼합하여 단계를 반복합니다. 각 NaCl 추출에서 뜨는은 신선한 플라스크로 전송됩니다. </li><li> 4 15 분에 대한 고속 (25,000 XG) 200 ML의 aliquots에 5 세포 extractions 및 원심 분리기에서 supernatants을 결합 ° C. 뜨는을 취소, 2 % 나트륨 hexametaphosphate 10 ML 각 세포 펠렛을 resuspend, 알약을 결합하고 2 % 나트륨 hexametaphosphate 200 ML에 볼륨을합니다. 30 분 상온에서 (150 RPM)에 의해 세포를 흔들어 씻으십시오. 이 단계의 끝에서 당신은 다음 단계로 진행하는 세포 중 하나만 튜브가 있어야합니다. </li><li> 4 15 분 ° C.에 대한 고속 (25,000 XG)에서 원심 분리기 TE (50 MM EDTA (에틸렌 다이아 민 테트라 초산)) 200 ML에 뜨는, resuspend 세포 펠렛을 무시, 10 분 상온에서 (150 RPM)에 의해 세포를 흔들어 씻는다. </li><li> 4 15 분에 대한 고속 (25,000 XG)에서 원심 분리기 ° C가, 15 ML TE (10 MM EDTA (에틸렌 다이아 민 테트라 초산))에 resuspend 뜨는을 폐기하고, -80에서 보관 200 μL aliquots ° C까지 DNA extractions 필요. </li></ol> 2. DNA 추출 및 정제 : <ol><li> 200 μL 5 M NaCl, 200 μL 10% SDS와 200 μL 미생물 세포 나누어지는 100 μL 14.3 M의 β - 메르 캅 토 에탄올을 추가합니다. 반전 4-6 배 부드럽게 섞는다. </li><li> 5 분 65 ° C 물 욕조에서 해동 다음 2 분에 대한 액화 질소에 잠수함이 잠수하여 동결 - 해동의 3 라운드에 따라 혼합물. 최종 10 분에 해동 연장. </li><li> 200 μL 5 M의 칼륨 아세테이트 (산도 5.5) 10 분 얼음에 장소를 추가합니다. 4 10 분 10,000 XG에 원심 분리기 ° C와 넓은 구멍 피펫 팁을 사용하여 새로운 튜브에 뜨는을 전송하기만하면됩니다. 1 시간을 위해 RNase 37과 부화는 ° C의 3 μL를 추가합니다. </li><li> 클로로포름의 동일한 음량을 추가, 역전에 의해 잠시 흔들하고, 실온에서 10 분 15,000 XG에 원심 분리기. </li><li> 신중하게 넓은 구멍 피펫 팁을 사용하여 새로운 튜브에 뜨는의 상단에 레이어를 전송합니다. 상단 및 하단 레이어 사이에있는 흰색 인터페이스 레이어를 pipetting하지 마십시오. 클로로포름 - 추출 절차를 반복합니다. </li><li> 냉장의 동일한 음량 (-20에서 ° C) 촉진의 DNA로 30 분 얼음에 뜨는와 부화 이소프로판올. 추가 </li><li> 4 10 분 최대 속도 (20,800 XG)에서 원심 분리기 ° C 펠렛 DNA 있습니다. </li><li> 냉장 1 ML 70 % 에탄올 (-20 ° C에서)로 뜨는 및 세척 DNA 펠렛 폐기하십시오. </li><li> 4 10 분 최대 속도 (20,800 XG) ° C에서 원심은, 표면에 뜨는를 버리고 10 분 70 %의 에탄올 세척 및 공기 건조 DNA 펠렛를 반복합니다. 70 % 에탄올 세척을 반복합니다. </li><li> 25 μL TE (10 MM EDTA (에틸렌 다이아 민 테트라 초산))에 Resuspend의 DNA, 65 ° 따뜻한 5 분 C는 여러 개의 튜브가 사용되었습니다면 한 튜브에 결합하고, TE 500 μL에 볼륨을 구성합니다. </li><li> 추가 500 μL 얼음처럼 차가운 (또는 4 ° C) 20 %의 폴리에틸렌 글리콜 (PEG) (1.2 M NaCl에서 준비). 펠렛 DNA 4 10 분 최대 속도로 10 분, 그리고 원심 분리기를위한 얼음에 품어 역전, ° C에 의해 부드럽게 섞는다. 제거하고 폐기 뜨는하고 냉장 70 % ethanol, 10 분 대한 항공 건기 1 mL로 pelleted DNA를 씻고, 그리고 30-50 μL TE or 분자 학년 물에 resuspend. 65 온난화에 의한 DNA의 resuspension을 촉진 ° -80시 5 분 및 저장 DNA에 대한 C ° C. </li></ol> 3. DNA 순도, 농도 및 크기 결정 : <ol><li> 280분의 260과 Nanodrop 1000 분광 광도계 또는 다른 적절한 장비를 사용하여 230분의 260 비율을 측정하여 DNA의 품질을 확인합니다. </li><li> 제조 업체의 지시에 따라 퀀트 - IT dsDNA 분석 키트를 사용하여 DNA 농도를 결정합니다. </li><li> 펄스 필드 겔 전기 영동을 사용하여 DNA의 크기를 결정합니다. 0.5X TBE와 1퍼센트 아가로 오스 겔을 준비하고 다음 주방장 매퍼 XA 시스템에 대한 매개 변수를 사용; 0.35 s의 초기 스위치 시간, 7.67 s의 마지막 스위치 시간, 120 °는 다음 위치에서 6.0 V / cm의 그라데이션에 대한 각도를 포함 리니어 R요소를 좋아지고. 30 분 1 X SYBR 그린 I와 젤 얼룩. 겔에 700-1000 NG 총 DNA 사이에로드합니다. 그림을 참조하십시오. 전체 프로토콜의 응축 형태 3. </li></ol> 대표 결과 : 세포 추출 : 순차 미생물 세포 추출물은 (supernatants) 추출물의 탁도가 extractions 증가의 숫자로 감소하는 것으로 나타났습니다. 이것은 각각의 연속된 세포 추출 다음과 같은 세포의 수를 감소를 제안합니다. 여기서주의해야할 중요 각 추가 셀 추출 단계는 오염 물질 도입에 대한 기회를 제공으로, 휴대 extractions의 번호가 최종 세포 펠렛은 전체 미생물 커뮤니티의 대표되도록 최소화해야한다는 것입니다. 오염의 다른 소스는 적절한 실험실 멸균 기술을 채택하여 제어했습니다. 예를 들어, 솔루션은 autoclaved DI 물의 준비와 필터를 소독했다. 컨테이너는 알코올로 소독 autoclaved하고, 자외선과 자외선 crosslinker에 따라 치료를했다. DNA 농도 및 품질 결정 : 프로토콜은 1.6 280분의 260 비율로 매트 샘플 g 당 HMW DNA (35-50킬로바이트) (그림 4)의 약 2 μg을 굴복하고, 0.7의 230분의 260 비율 (표 1). 230분의 260 비율이 같은 rRNA 유전자는 별도의 연구 7 관찰했던 16의 PCR - 증폭 등 하류 분자 기반 응용 프로그램에 대한 억제 낮은하지 나타나지만. EPS와 소금, hypersaline 매트에서 해당 DNA 오염의 두 가지 주요 출처가에 유의하는 것이 중요합니다. 그것은 이러한 오염 물질의 흔적은 다운 스트림 애플 리케이션에 미치는 영향을 줄이기 위해 엄청난 노력에도 불구하고 230분의 260 비율에 영향을 미치는있을 수 있으므로 가능합니다. DNA 크기 결정 : 펄스 필드 겔 전기 영동은 그림 3과 같이 DNA의 얼룩의 발생 간헐적인 방향 스위치와 pulsating 전류 흐름을 사용합니다. 우리 프로토콜은 약 35-50킬로바이트의 H​​MW DNA를 나왔고. 일부 DNA 크기가 30킬로바이트보다 작아야 수 있지만, 그것은 fosmid 복제가 ~ 40킬로바이트 DNA 삽입이 필요하며 큰 DNA 조각은 그대로 biosynthetic 경로에 큰 액세스를 제공하는 주어진 35킬로바이트 위의 얼룩의 일부를 가지고하는 것이 중요합니다. 우리의 연구에, 35킬로바이트 위의 DNA의 얼룩은 excised되었으며 대형 삽입 벡터 클로닝 및 기타 분자 애플 리케이션을위한 정화. <img src="/files/ftp_upload/2887/2887fig1.jpg" alt="그림 1"/> 엘류테라, 바하마에있는 그림 1. Hypersaline의 미생물 매트 샘플링 사이트 (빅 폰드). <img src="/files/ftp_upload/2887/2887fig2.jpg" alt="그림 2"/> 그림 2. 본 연구에서 사용되는 hypersaline 미생물 매트의 단면. 미생물 매트는 엘류테라, 바하마에있는 hypersaline 연못로부터 얻은 것입니다. <img src="/files/ftp_upload/2887/2887fig3.jpg" alt="그림 3"/> 그림 3. 미생물의 세포 추출, 세포 용해, 추출, metagenomic DNA의 정화에 참여 절차 도식 표현은. <a href="http://www.jove.com/files/ftp_upload/2887/2887fig3large.jpg">큰 이미지를 보려면 여기를 클릭하십시오.</a> <img src="/files/ftp_upload/2887/2887fig4.jpg" alt="그림 4"/> 그림 4. 펄스 필드 겔 전기 영동을 사용하여 추출한 DNA의 분자량 특성화. 레인 M은 HMW 마커이며, 골목길 1과 2는 위의 프로토콜을 사용 엘류테라 hypersaline 매트에서 추출한 DNA의 복제 metagenomic 있습니다. <table border="1"><tbody><tr><td> 추출 방법 </td><td></td><td> 농도 NG / G </td><td> 280분의 260 </td><td> 230분의 260 </td></tr><tr><td> PEG </td><td> 담당자 1 </td><td> 1806.1 </td><td> 1.64 </td><td> 0.76 </td></tr><tr><td></td><td> 담당자 2 </td><td> 2010.8 </td><td> 1.61 </td><td> 0.75 </td></tr></tbody></table> 표 1. DNA의 농도 및 품질 측정은 미생물의 hypersaline 매트에서 추출한.

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

관심 없음 충돌 선언하지 않습니다.

복잡하고 매우 다양한 미생물 매트 샘플에서 전체 세포 제거 실용적 아니라는 것을 감안할 때, 기본 우려가 추출된 세포가 전체 미생물 매트 커뮤니티를 대표하는 얼마나 잘됩니다. 이전 연구에서는 미생물의 유전자 16 rRNA의 PCR - DGGE 분석이 프로토콜에 사용되는 다섯 셀 제거 단계를 전체 미생물 매트 커뮤니티 7 대표하는 세포를 추출 것으로 나타났다. 세포 추출의 실제 번호는 전체 미?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> β - 메르 캅 토 에탄올 </td><td> 시그마 - 알드리치 </td><td> M3148 </td><td></td></tr><tr><td> 폴리에틸렌 글리콜 8000 </td><td> Promega </td><td> V3011 </td><td> 1.2 M NaCl에 최고 20 % </td></tr><tr><td> 칼륨 아세테이트 </td><td> 피셔 사이 언티픽 </td><td></td><td> 피셔 사이 언티픽 </td></tr><tr><td> 퀀트 - IT dsDNA 분석 키트 </td><td> Invitrogen </td><td> Q33130 </td><td></td></tr><tr><td> RNase </td><td> Epicentre </td><td> MRNA092 </td><td></td></tr><tr><td> 나트륨 염화물 </td><td> BDH 화학 </td><td> BDH8014 </td><td> 적절한 CONC. </td></tr><tr><td> 나트륨 Dodecyl의 황산 </td><td> 피셔 사이 언티픽 </td><td> 03-500-509 </td><td> 물에서 10 % </td></tr><tr><td> 나트륨 hexametaphosphate </td><td> EMD 화학 </td><td> SX0583 - 3 </td><td> 물에 2% </td></tr><tr><td> TBE </td><td> 피셔 사이 언티픽 </td><td> BP1333 - 1 </td><td></td></tr><tr><td> 요리사 매퍼 XA 시스템 </td><td> 바이오 래드 연구소 </td><td> 170-3670 </td><td></td></tr><tr><td> NanoDrop 1000 분광 광도계 </td><td> 써모 과학 </td><td> ND - 1000 </td><td></td></tr><tr><td> Vortexer </td><td> 과학 산업 주식 회사 </td><td></td><td></td></tr><tr><td> 자외선 Crosslinker </td><td> UVP </td><td></td><td></td></tr><tr><td> 워링 믹서기 </td><td> 워링 실험실 </td><td> LB10S </td><td></td></tr></tbody></table>

extraction of high molecular weight dna from microbial mats

metagenomic 데이터의 성공적인 정확한 분석과 해석은 고품질, 높은 분자량 (HMW) 커뮤니티 DNA의 추출 효율에 의존적일 수 밖에 없습니다. 그러나, 환경 매트 샘플은 종종 높은 품질 HMW DNA의 큰 농도를 구하기 위해 어려움을 제기. Hypersaline의 미생물 매트 높은 세포외 고분자 물질의 양의 (EPS) 1 추출한 DNA의 다운 스트림 애플 리케이션을 억제 수 염분이 포함되어 있습니다. 직접적이고 거친 방법은 자주 내화물 샘플에서 DNA 추출에 사용됩니다. 매트, 접착제 매트릭스에 EPS 직접 용해하는 동안 DNA 2,3를 바인딩하기 때문에 이러한 방법은 일반적으로 사용됩니다. 엄격한 추출 방법의 결과로, DNA되고 작은 크기 4,5,6으로 조각. DNA 따라서 대형 삽입 벡터 복제에 대한 부적 절한됩니다. 이러한 제한을 우회하기 위해, 우리는 hypersaline 미생물 매트에서 좋은 품질과 수량의 HMW DNA를 추출하는 개선된 방법을보고합니다. 우리는 혼합 및 차동 원심 분리를 통해 배경 매트 매트릭스에서 미생물 세포의 분리와 관련된 간접적인 방법을 고용. 기계적 및 화학적 절차 조합이 추출된 미생물 세포에서 DNA를 추출하고 정화하는 데 사용되었다. 우리 프로토콜 1.6 280분의 260의 비율로, 매트 샘플 g 당 HMW DNA (35~50킬로바이트)의 약 2 μg을 산출. 또한, 16 rRNA 유전자 7 증폭이 프로토콜은 오염 물질의 억제 효과를 최소화하거나 제거할 수 있다고 제안합니다. 우리의 결과는 기능 metagenomic 연구 미생물 매트에서 HMW의 DNA의 추출을위한 적절한 방법을 제공하고 DNA 추출이 어려운있는에서 다른 환경 시료에 적용할 수 있습니다.

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

Extraction of High Molecular Weight DNA from Microbial Mats

우리는 hypersaline 미생물 매트에서 높은 분자량의 DNA를 추출하기위한 개선된 프로토콜을 제공합니다. 미생물 세포는 DNA 추출 및 정화하기 전에 매트 매트릭스에서 분리됩니다. 이것은 농도, 품질 및 DNA의 크기를 향상시킵니다. 프로토콜은 다른 내화물 샘플에 사용할 수 있습니다.

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

Research

JoVE Journal

Biology

19.4K 조회수.  Arnold School of Public Health, University of South Carolina. 우리는 hypersaline 미생물 매트에서 높은 분자량의 DNA를 추출하기위한 개선된 프로토콜을 제공합니다. 미생물 세포는 DNA 추출 및 정화하기 전에 매트 매트릭스에서 분리됩니다. 이것은 농도, 품질 및 DNA의 크기를 향상시킵니다. 프로토콜은 다른 내화물 샘플에 사용할 수 있습니다.

비디오: Extraction of High Molecular Weight DNA from Microbial Mats

Biology of Microbial Communities  - Interview

미생물 커뮤니티의 생물학 - 인터뷰

연구

생물학

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

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

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

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

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 ...

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 ...

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 ...

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 ...

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

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

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

점액 아가로 오스 겔 전기 영동 : 높은 분자량 당 단백질에 대한 웨스턴 블로 팅


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

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

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

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

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 ...

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

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

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

고처리량 DNA 추출 플레이트의 로딩을 위한 수정된 방법은 오염 가능성을 감소시니다.

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

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

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

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

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

Ca2+ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

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

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

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

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

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

Caenorhabditis elegans Genomic DNA의 소규모 추출

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