우리는 표본을 얻기에 도움 앨런 Tasker (USDA-진딧물, Riverdale, MD), 스티븐 콤튼 (클렘슨), 셰리 Aultman (클렘슨), 크레이그 램지 (USDA-진딧물, 포트 콜린스, 콜로라도), 그리고 베티 Marose (UMD)를 감사 . 우리는 비디오의 촬영에 그의 작업에 대한 학생 앤드류 아드리안 (UA-헌츠빌)와 데릭 대커 (UA-헌츠빌)이 프로토콜을 테스트하고 그들의 도움, 그리고 요셉 Herdy 감사드립니다. 이 작품은 국립 수산 및 야생 동물 재단에 의해 후원되었다.

<ol>
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U.S.A. 102 (23), 8369-8374 (2005).</li><li>Roodt-Wilding, R., Spies, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+relationships+in+southern+African+chloridoid+grasses+(Poaceae)+based+on+nuclear+and+chloroplast+sequence+data.">Phylogenetic relationships in southern African chloridoid grasses (Poaceae) based on nuclear and chloroplast sequence data.</a> Systematics and Biodiversity. 4, 401-415 (2006).</li><li>Brody, J. R., Kern, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+boric+acid:+a+Tris-free,+cooler+conductive+medium+for+DNA+electrophoresis.">Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis.</a> BioTechniques. 36, 214-216 (2004).</li><li>Chou, C. -. H., Tsai, C. 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우리는 공개 할게 없다.

<html> 미국의 보육 및 조경 산업은 이국적이고 새로운 식물 종을 재배하고 판매에 번성. 무역의 성장 세계화의 결합 이것은, 침입 식물 종 (种)을 입력 수립, 그리고 미국에서 연방 정부가 침략이 될 수있는 가능성을 포함하여 자주 사용할 수없는 정보에 의존 그러한 식물을 조절하는 능력을 확산 것이라는 가능성을 증가 , 토종과 귀화 taxa의 올바른 분류법, 그리고 유전 distinctness. 침입 식물이 우리의 지식은 종종 제한이기 때문에, 숨겨진 침입 특성과 수입 식물은 기꺼이 그들만이 우리 농업과 자연 자원을 침해하는 나중에 배우고 도입되었습니다. 이 프로토콜은 저와 관련된 이러한 문제를 해결하는 것을 목표로 정확하게 야생 형 cogongrass과 관상용 JBG의 대응의 복귀 형태를 구분할 수있는 최초의 단순 분자 방법을 제공하여 cylindrica 품종. 이 프로토콜의 개발을위한 jove_content &quot;&gt;은 야생 형 cogongrass은 2008 년 6 월 제이 플로리다 인근 산타 로사 카운티의 연못 크릭 임업 단위에서 귀화한 인구로부터 수집되었다. JBG는 상업 탁아소 (블루버드 보육, INC로부터 조달되었다 .. Riverdale, MA의 대학 공원 일부터 6 월 2009 년;) 6 월 2008 년뿐만 아니라 콜롬비아에서 주택 소유자의 컬렉션에서 미주리 JBG 준 2008 년 클렘슨 대학, 사우스 캐롤라이나에서 캠벨 지질 박물관의 안뜰에서 얻은 있었을갑니다 및 콜롬비아, 2009 년 미주리 (리랜드 Cseke 식별)의 주택 소유자의 정원에서. 모든 식물은 헌츠빌에서 알라바마의 대학 (헌츠빌, AL)에있는 온실에서 유지되었다. 이들 식물에서 수집한 DNA의 유전자 시퀀싱은 일반적으로 바코드 식물 2로 사용 9 독립적인 DNA 영역의 깊이 비교에 포함되어있었습니다. 모든 경우에서, JBG의 시퀀스를 따라서하는 데 도움 JBG 되돌릴 분들에게 100 % 일치했다JBG 실제로 녹색, 침입 형태로 되돌리려는 것을 확인합니다. 오직 핵 ITS와 엽록체 trnL-F 영역은 유전자 cogongrass와 JBG 구별하는 데 사용할 수있는 차이점이 있습니다. trnL-F 지역은 2 SNPs 2 InDels합니다 (삽입 및 삭제)이있는 동안은 ITS 지역, cogongrass와 JBG 사이 세 SNPs (단일 염기 polymorphisms)의 총 있습니다. 이러한 유전적 차이는 야생 형 cogongrass와 JBG이갑니다 구분 수있는 개발되는 다양한 특정 PCR의 primers를 허용했다. 가장 안정적인 결과는 plastid trnL-F 지역에서 파생된 primers에서 왔습니다. 따라서이 프로토콜은 cogongrass, JBG의 엽록체의 게놈의 trnL-F 지역 및 JBG (그림 4) 되돌 간의 순서의 차이에 따라 달라집니다. 문제의 종류, 더 구체적인 primers를 생성할 수 있도록하고 밀접하게 관련 종, 야외에서 잘못된 반응을 방지하기 위해I.의 리터 알려져 trnL-F 시퀀스 cylindrica 품종은 관련 잔디 종 (29 종 43 독립적인 시퀀스, 예를 들어, Cymbopogon citratus, Sorghastrum incompletum, Coix lacryma-jobi, Miscanthus sinensis의, Saccharum officinarum, 수수 halepense)에서 trnL-F 시퀀스와 비교되었다. 우리는 잔디, 다양한 특정 primers의 특이성의 29 여종에 걸쳐 다양한 전용 프라이머 시퀀스를 검사 있지만 종합적으로 대부분의 다른 잔디 종에서 DNA를 증폭하는 기능에 대해 검사되지 않았습니다. 따라서 DNA 추출에 사용되는 조직은 신중하게 나로 식별되어야합니다 cylindrica 전에이 프로토콜을 시작합니다. 잔디는 다음 cogongrass 또는 JBG 중으로 확인할 수 없다면 우리는 시퀀스 cogongrass 또는 JBG에 정확히 일치하는 것을 확인하기 위해 PCR 제품을 시퀀싱하는 것이 좋습니다. 현재 주어진 잔디 시편의 신원을 확인하는 가장 정확한 방법은 t 모두에서 PCR을 수행하는 것입니다rnL-F와 ITS 지역 시퀀스 PCR 제품을 확인하고 정확하게 식별 taxa에서 알려진 시퀀스의 시퀀스를 비교하여 따라갔다. DNA가 컨트롤이 프로토콜에 설명된 primers (trnL-F 지역의 경우) 또는 다른 primers 기타 출판물 13-15에서 해당 이용할 수를 사용하여 증폭 할 수 있습니다. 시퀀싱이 훨씬 노동 및 간소화된 절차를 사용하여보다 집중적인 비용입니다. PCR에 사용된 primers의 품질은 프로 시저의 성공에 중요합니다. 우리는 경사 바이오 주식 회사 (에서 제공하는이 절차에 대한 primers를 만들었 <a href="http://www.obliquebio.com/web/" target="_blank">http://www.obliquebio.com/web/</a> , 헌츠빌, AL). 경사 바이오에서 primers 주문에 대한 장점은 그들이 우리 실험실에서 최적화에 사용된 primers와 같은 동일한 샘플 생산 시리즈에서 각 프라이머의 aliquots 다수를 저장한다는 것입니다. 따라서, primers는 같은 순서를 가지고 있지만, t 없습니다야이 프로토콜에 사용되었습니다 동일한 생산 많이에서 왔어요. 같은 많은로부터 primers를 사용하여 PCR의 primers의 품질의 차이에서 발생할 수있는 절차에 필요없는 변수를 방지하는 데 도움이됩니다. 마찬가지로 다른 DNA 형성 촉매의 polymerases는 PCR, 사용된 DNA 형성 촉매 효소의 품질을 위해 좋을 동안 PCR 결과의 품질에 영향을 미칠 것이다. PCR 시약의 더 나은 일관성을 허용하기 위해, 우리는 Clontech의 시약을 사용하여 프로토콜을 최적화했습니다. 장점이 효소는 (Clontech, 캘리포니아 주 마운틴 뷰, 고양이 # 639201 또는 639202) 강력한, 핫 스타트 DNA 형성 촉매의 중합 효소의 혼합물과 높은 특이성과보다 정확한 amplifications를 제공하는 데 도움이 증명 독서 효소이다. 이 프로토콜은 maternally 목초에서 상속되는 엽록체의 DNA에 의존하기 때문에 cogongrass 및 JBG의 genotypes 사이의 하이브리드화 이벤트는 우리의 분자 식별 절차에 캡처되지 않을 수 있습니다. hypridization가 suspe는 경우cted, 우리는 양쪽 부모로부터 상속됩니다 핵 영역을 사용하는 것이 좋습니다. 식물 genotyping에서 고려해야 할 가장 일반적으로 사용되는 비 plastid 변수 영역은 핵 ribosomal ITS 지역 13-15,17입니다. 현재, 우리는 동일한 PCR 튜브에 핵 ITS 영역의와 엽록체 trnL-F 영역의 증폭을 멀티플렉싱으로 진전된다. 핵 DNA 영역으로 plastid 멀티플렉싱하는 것은 잠재적으로 홀로 중 하나를 사용하는 한계를 우회할 것이다 있지만, 이러한 방법은 케이스별로 사건에 타당성을 결정하는 최적화 및 추가적인 평가가 필요합니다. 정량 실시간 PCR (qPCR)과 같은 분자 비콘 (형광 프라이머 프로브) 등 최신 기술의 사용은 또한 고장 안전하고 정확한 식물 genotyping 방법으로 취급되고있다. 여기에 제시된 프로토콜은 JBG는 야생 형 cogongrass의에서 되돌릴 구별하는 빠르고 안정적​​인 접근 방식을 제공합니다. 우리는 사용 권장이 프로토콜의 RS는이 프로토콜의 사용에서 파생된 결과를보고하기 위해 저희에게 연락하십시오. 이러한 공유 정보 JBG는 되돌려의 분포에 관한 정보를 제공하는 데 도움이됩니다. 이것은 USDA의 규제가 매우 침략적 cogongrass 품종의 보급과 잠재 하이브 리다이 제이션을 회피 할 수있는 행동에 객관적인 판단을 내릴 도움이 될 것입니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> DNeasy 식물 미니 키트 </td><td> Qiagen, 발렌시아, 캘리포니아 </td><td> 69,104 또는 69,106 </td><td> 양질의 DNA를 얻을 모든 평판 게놈 공장의 DNA 키트는 다음 절차에 대해 잘 동작한다. </td></tr><tr><td> trnF (GAA)-F </td><td> 경사 바이오 주식 회사 </td><td> 3-0578 </td><td> 앞으로 긍정적인 제어 입문서 </td></tr><tr><td> trnL (5 &#39;엑손)-C </td><td> 경사 바이오 주식 회사 </td><td> 3-0579 </td><td> 긍정적인 컨트롤 프라이머를 역방향 </td></tr><tr><td> trnLF-C-F1 </td><td> 경사 바이오 주식 회사 </td><td> 3-0864 </td><td> 앞으로 야생 형 cogongrass의 프라이머 </td></tr><tr><td> trnLF-C-R1 </td><td> 경사 바이오 주식 회사 </td><td> 3-0865 </td><td> 개머리판야생 형 cogongrass의 프라이머 </td></tr><tr><td> trnLF-R-F2 </td><td> 경사 바이오 주식 회사 </td><td> 3-0866 </td><td> 앞으로 JBG와 프라이머를 되돌 JBG </td></tr><tr><td> trnLF-R-R2 </td><td> 경사 바이오 주식 회사 </td><td> 3-0867 </td><td> JBG와 프라이머를 되돌 JBG를 바꾸게되면 </td></tr><tr><td> 장점 UltraPure PCR dNTP 믹스 </td><td> Clontech, 캘리포니아 주 마운틴 뷰 </td><td> 639,125 </td><td></td></tr><tr><td> 장점이 중합 효소 </td><td> Clontech, 캘리포니아 주 마운틴 뷰 </td><td> 639,201 또는 639,202 </td><td> 좋은 증거 독서, 핫 스타트 DNA 형성 촉매의 중합 효소 </td></tr></tbody></table>

a pcr-based genotyping method to distinguish between wild-type and ornamental varieties of imperata cylindrica

야생 형 저 cylindrica (cogongrass)는 부정적인 아프리카, 아시아, 유럽, 뉴질랜드, 오세아니아 및 미주 1-2에 걸쳐 73 개국에서 농업과 천연 자원에 영향을 미치는, 세계 10 대 최악의 침입 식물 중 하나입니다. Cogongrass는 토착 식물 종의 큰 다양성을 치환하고 차례로 마초와 쉼터를위한 전이 원시 식물 수종에 따라 야생 동물을 위협 빠르게 - 확산, monodominant 스탠드를 형성합니다. 문제를 추가하려면, 관상용 다양한 [나 cylindrica VAR. koenigii (Retzius가)] 널리 Imperata cylindrica &#39;Rubra&#39;, 레드 바론, 일본 혈액 풀 (JBG)의 이름하에 판매됩니다. 이것은 다양한 putatively 살균 및 비침 투이며 붉은 단풍을위한 바람직한 관상용으로 간주됩니다. 그러나, 정확한 조건 하에서, JBG가 가능한 종자를 생산할 수있다 (캐롤 Holko 2009 개인 통신)와 나는 녹색으로 되돌릴 수 있습니다그것이 야생 형 침략 다양한 4 (그림 1)의 구분되는 특징에 걸릴 cogongrass에서 종종 구별할 수있다 nvasive 형식입니다. 이것은 신분도 잘받은 식물 taxonomists 위해 형태학 어려운 작업을 사용하여 만듭니다. 공격적인 녹색 표현형에 JBG의 복귀도 드문 아닙니다. 코딩 및 원자력과 엽록체 둘 다 DNA의 변수 영역의 시퀀스 비교를 사용하여, 우리는 JBG는 메릴랜드, 사우스 캐롤라이나, 그리고 미주리 주에서 내에 녹색 침략으로 되돌 것으로 확인되었습니다. 활성 cogongrass의 침입이 아닌 곳에 JBG는 대륙 미국에서 거의 모든 국가에서 판매하고 심은되었습니다. 잘 이해되지에서는 되돌릴 문제의 범위는 되돌 식물 문서화하고 종종 파괴 때문이다. 이 분자 프로토콜의 응용 JBG가갑니다 식별할 수있는 방법을 제공하고 공동 발생이 품종을 유지하는 데 도움ND 가능성이 hybridizing. Cogongrass가 다른 유전자형으로 건널 때 고맙게 여기게 outcrosser이며, - 바람의 분산 가능한 생산 5-7 넓은 거리를 통해 cogongrass 뿌리 씨앗을하실 수 있습니다. JBG는 cogongrass보다 약간 다른 유전자형을 가지고 있으며, cogongrass으로 가능한 하이브리드를 구성할 수 있습니다. 문제를 추가하려면, JBG는 cogongrass 8-10보다 추위와 관용 그늘이고,이 두 품종 간의 유전자 흐름은 더 공격적이다 하이브리드를 생성할 가능성이 야생 형 cogongrass보다 성의 관용, 그리고 차가운 그늘. 야생 형 cogongrass 현재 동남 미국에서 전세계 100 만 490여 헥타르, 감염 시켰어 있지만 그것은 500,000헥타르 통해 감염 시켰어하고 빠르게 북쪽의 광범위한 틈새 시장 및 지리적 잠재 3,7,11에 의한 확산으로 미국의 대부분을 차지하는이 가능합니다. 유전자 건널목의 잠재력은 USDA-진딧물 연방 유해 주 프로그램에 대한 심각한 우려입니다. 현재 USDA-진딧물은 주와 무에서 JBG 금지오히려 주요 cogongrass의 infestations (예, 플로리다, 알라바마, 미시시피)가 있습니다. cogongrass와 JBG 자신의 배포판을 확장할 그러나 결합에서 두 품종을 방지하는 것은 더 어려울 수 있습니다. 또한, JBG 되돌릴 수의 배포는 현재 알려지지 조직 형태를 통해 이러한 품종을 식별하는 능력없이 일부 cogongrass의 infestations은 JBG는 되돌려의 결과가 될 수도 있습니다. 불행히도, 신분의 현재 분자 방법은 일반적으로 소모 및 비용 시간은 둘 다 AFLP (증폭 단편 길이 Polymorphisms)와 DNA 시퀀싱에 의존하고 있습니다. 여기서는 정확 cogongrass와 JBG 되돌릴 구별하는 최초의 비용 절감 효과 및 안정적인 PCR 기반의 분자 genotyping 방법을 제시한다.

Watch this Scientific Journal Video about A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica at JoVE.com

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

우리는 엽록체 trnL-F 스페이서 지역 내의 대상의 DNA 서열 차이의 종류를 구분하는 것이 다양한 특정 PCR primers의 직원을 고용하고 비용 효과적이고 신속한 분자 genotyping 프로토콜을 제공<em&gt; Imperata cylindrica</em&gt; (cogongrass) 혼자 형태로 구분할 수없는. 이러한 종류의 연방 목록에 유해 잡초, cogongrass과 밀접하게 관련, 넓은 확산 장식 다양성, 내가 포함<em&gt;. cylindrica</em&gt; VAR.<em&gt; koenigii</em&gt; (일본어 혈액 풀).

의 야생 - 타입과 장식 품종을 구별하기 PCR 기반 Genotyping 방법 Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

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JoVE Journal

Biology

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

31.4K 조회수.  The University of Alabama, Huntsville. 우리는 엽록체 trnL-F 스페이서 지역 내의 대상의 DNA 서열 차이의 종류를 구분하는 것이 다양한 특정 PCR primers의 직원을 고용하고 비용 효과적이고 신속한 분자 genotyping 프로토콜을 제공 Imperata cylindrica (cogongrass) 혼자 형태로 구분할 수없는. 이러한 종류의 연방 목록에 유해 잡초, cogongrass과 밀접하게 관련, 넓은 확산 장식 다양성, 내가 포함. cylin...

비디오: A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing : 정확한 Genotyping을위한 간단한 방법

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

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Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

내생 와일드 타입 BRCA1을 표현 세포를 사용하는 동종 재조합에 BRCA1 변이의 효과를 확인

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

단백질 막 중첩 분석 : 가용성식이 및 불용성 단백질 간의 상호 작용을 테스트하는 프로토콜 체외에서</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

Mycoplasma의 최적화된 PCR 기반의 감지

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

얼음 총액 : 성장을위한 방법 Arabidopsis높은 처리량 Genotyping을위한 96 - 웰 플레이트에와 토마토 식물

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

이전 DNA의 정제없이 식물과 동물 샘플의 Genotyping

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

대규모 SNP 유전자형 응용 프로그램에 대한 Infinium 분석

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

고해상도 용융 분석과 PCR을 이용하여 신속하고 효율적인 Zebrafish의 유전자형

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag 분석 - 높은 처리량, Sc2.0 유전형에 대한 실시간 PCR 분석

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

야생형과 간암의 시알 산의 결정<em&gt; CMAH</em&gt; 넉 아웃 마우스.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...