리처드 W. 베린저와 데이비드 L. 애쉬, 기술 지원 및 촬영에 대한 응용 과학자, 써모 사이 언티픽 NanoDrop 제품.

1. NanoDrop 2000c 분광을 사용 Microvolume 핵산 부량 <ol><li> 시작하려면 아래 광학 표면에 깨끗한 탈이온수 2 ~ 3 μL를 pipetting하여 microvolume 분광 광도계 샘플 유지 시스템의 위턱과 아래턱에 광학 표면을 청소하십시오. </li><li> 상단 받침대는 탈이온수 접촉 들어오는 있도록, 레버 암을 닫습니다. 레버 암을 들어 깨끗하고, 건조, 보푸라기가없는 실험실 닦아와 함께 광학 표면을 모두 닦아. </li><li> NanoDrop 소프트웨어를 열고 핵산 응용 프로그램을 선택합니다. 낮은 광학 표면에 버퍼 1 μL를 분배하여 빈 측정을 수행하는 작은 볼륨 보정 pipettor을 사용하십시오. 레버 암을 절감하고 핵산 응용 프로그램에서 &quot;공백&quot;을 선택하십시오. </li><li> 일단 빈 측정이 완료되면, 깨끗하고, 건조, 보푸라기가없는 실험실 닦아와 함께 광학 표면 모두를 청소하십시오. </li><li> 측정되어야하는 예제에 해당하는 상수를 선택합니다. <table border="1"><tbody><tr><td> 샘플 유형 </td><td> 옵션 선택 </td><td> 농도를 계산하는 데 사용 상수 </td></tr><tr><td> dsDNA </td><td> DNA - 50 </td><td> 50 </td></tr><tr><td> ssDNA </td><td> DNA - 33 </td><td> 33 </td></tr><tr><td> RNA </td><td> RNA - 40 </td><td> 40 </td></tr><tr><td> Oligo </td><td> 사용자 정의 </td><td> 15-150 </td></tr></tbody></table> 표 1. NanoDrop microvolume 분광 광도계, TrayCell microcell 쿠베트와 함께 사용 전통 쿠베트 기반 분광 광도계 및 PicoGreen 분석과 함께 microvolume의 NanoDrop의 fluorospectrometer를 사용하여 직접 A280 흡광도 측정에 대한 일반적인 핵산 농도 범위. </li><li> 낮은 광학 받침대에 핵산 샘플의 1 μL를 분배하고 레버 암을 닫습니다. 측정 독립적인 볼륨이기 때문에 예제는 만들 수있는 측정을위한 두 광학 표면 사이의 격차에 다리를 놓으려고해야합니다. </li><li> 어플 리케이션 소프트웨어에서 &quot;법안&quot;를 선택하십시오. 소프트웨어는 자동 핵산 농도 및 순도 비율을 계산합니다. 샘플 측정에 따라, 스펙트럼 출력을 검토합니다. </li><li> 소프트웨어는 자동 핵산 농도 및 순도 비율을 계산합니다. 샘플 측정에 따라 샘플의 품질을 평가하기 위해 스펙트럼 이미지를 검토합니다. </li><li> 전형적인 핵산 샘플은 매우 특징적인 프로필을 것입니다. <img src="/files/ftp_upload/2565/2565fig1.jpg" alt="그림 1" /> 그림 1. 전형적인 핵산 샘플은 매우 독특한 프로필을 것입니다. </li><li> 특정 핵산 분리 기술과 관련된 오염 물질의 일반적인 소스는 페놀 / Trizol 및 열 추출을 포함합니다. 페놀 / Trizol 추출의 경우, 잔여 시약의 오염은 220-240 nm의 사이에 비정상적인 스펙트럼에 의해뿐만 아니라 260-280 nm의 영역에 교대로 표시됩니다. 반대로 열 추출에서 잔여 구아니딘 230 nm의 근처에 최대 230 nm의 약 240 nm의에 여물 변화에 기여할 수 있습니다. <img src="/files/ftp_upload/2565/2565fig2.jpg" alt="그림 2" /> 그림 2. 샘플 B와 C의 봉우리와 골짜기에있는 교대는이 오염 물질은 핵산 샘플의 스펙트럼에 영향을 미칠 수있는 방법을 설명 샘플을 비교했다. </li><li> 정확하게 샘플의 품질을 평가하기 위해, 280분의 260 또는 230분의 260 비율이 전체 스펙트럼 품질와 함께 분석해야합니다. 순수한 핵산은 일반적으로 각각 ~ 1.8 280분의 260 비율과 DNA와 RNA에 대한 ~ 2.0 280분의 260 비율을 얻을 수 있습니다. 이 비율은 산도와 빈과 샘플 측정하기 위해 사용되는 버퍼의 이온 강도에 따라 좌우됩니다. 기본 솔루션이 0.2-0.3의 비율을 통해 - 대표되지만 산성 솔루션은 0.2-0.3의 비율을 세 이하 대표 것입니다. 크게 다른 순도 비율 280 nm의 또는 근처에 강하게 흡수 단백질, 페놀 또는 기타 오염 물질의 존재를 나타낼 수 있습니다. 230분의 260 순도 비율은 1.8-2.2의 범위에서 일반적으로 &quot;순수&quot;핵산의 산성에 대한 값으로 DNA 순도의 두 번째 측정하기위한 한 방법입니다. 예상 값보다 훨씬 낮은 순도의 비율이 사용되는 절연 기술 추가 최적화가 필요할 수 있습니다 나타낼 수 있습니다. </li><li> NanoDrop spectrophotometers도 직접 흡광도를 사용하여 단백질이나 단백질 colorimetric assays로를 계량하는 데 사용할 수 있습니다. 적절한 열 형성과 재현성을 보장하기 위해, 2 μl 샘플은 단백질 샘플하시기 바랍니다. 다음 조브 비디오를 참조하십시오 NanoDrop 분광 사용 Microvolume 단백질 농도 결정 <a href="http://www.jove.com/index/Details.stp?ID=1610">http://www.jove.com/index/Details.stp?ID=1610을</a> </li></ol><ol><li> NanoDrop 3300을 사용하여 높은 감도 microvolume 핵산 quantitation을 발휘하려면 PicoGreen 형광 키트가 사용됩니다. 시작하려면, 상온에 키트 표준 및 모든 핵산 샘플을 평형. 형광 샘플 빛을 민감한 있으며 황색 또는 알루미늄 포장 튜브에 보관해야합니다. </li><li> 필요하게 될 PicoGreen 작업 솔루션을뿐만 아니라 측정하는 모든 표준 및 샘플에 대한 충분한 1X TE 버퍼를 준비합니다. 제조 업체의 절차에 따라 nuclease 무료 물을 제공 20x TE 버퍼를 diluting하여 1xTE 버퍼를 준비합니다. 반응 볼륨은 200 μL로부터 거의 10 UL 범위 수 있습니다. 이 예제에서, 10 μL 반응 볼륨이 준비됩니다. </li><li> nuclease 무료 튜브 또는 튜브에 배 최종 원하는 농도에 순차적으로 희석 dsDNA 표준을 준비합니다. nuclease 무료 호박이나 박 덮인 튜브 라벨 개별로 희석 dsDNA 기준의 각 5 μL를 전송합니다. </li><li> 적절하게 표시 nuclease - 무료 호박이나 박 덮인 튜브에 관심 각각의 dsDNA 샘플의 나누어지는 5 μL. </li><li> 제조 업체의 프로토콜에 따라 PicoGreen 작업 솔루션을 준비합니다. 관심의 표준 또는 샘플 (이 예에서는 5 μL) 중 하나를 포함하는 각각의 튜브에 PicoGreen 작업 솔루션의 동일한 볼륨을 전송합니다. </li><li> 참조 솔루션으로 제공하는 대조군을 준비하는 1X TE 버퍼와 PicoGreen 작업 솔루션의 동등 권을 결합합니다. </li><li> 부드러운 pipetting에 의해 철저하게 각각의 반응 관의 내용을 섞어 5 분 실온에서 반응을 품어. 형광 신호가 온도의 영향으로 모든 기준과 샘플을 측정하기 전에 동일한 온도 equilibrated해야합니다. </li><li> 악기를 준비하고 측정을 수행하려면 먼저 샘플 유지 시스템의 낮은과 상부 표면을 청소하십시오. 낮은 광학 표면에 탈이온수의 Pipet 2-3 μL가 가까이 후 레버 팔을 열고 깨끗하고 보푸라기가 무료로 상단과 하단 받침대를 얼룩, 연구실 닦으십시오. 표면은 보풀이 여기 소스의 존재에 형광 전시하고 측정을 방해할 수로 진행하기 전에 보푸라기가 무료입니다 보장합니다. </li><li> , 악기 소프트웨어를 열고 &quot;핵산&quot;응용 프로그램을 선택하고 &quot;PicoGreen - dsDNA&quot;옵션을 선택합니다. </li><li> 악기를 공백으로, 하단 받침대에 1X TE 2 μL를 추가 팔을 가까이하고 악기 소프트웨어 분야에서 &quot;공백&quot;을 선택하십시오. 일단 비어가 완료, 샘플 유지 시스템의 두 표면에서 빈 솔루션을 얼룩. </li><li> 부드러운 pipetting하여 레퍼런스 솔루션을 섞는다. 하단 받침대에 낮은 유지 팁, 피펫 2 μL를 사용하고 팔을 닫습니다. </li><li> 측정 타입의 &quot;참조&quot;를 선택하고 원하는 단위를 선택합니다. 측정주기를 시작하려면 &quot;법안&quot;을 클릭하십시오. </li><li> 측정주기가 완료되면, 팔을 열고 철저하게 샘플 보존 시스템 표면을 얼룩. 다섯 최대 측정하면 각 복제에 대한 새로운 나누어지는을 사용하여 참조 복제합니다. </li><li> 표준 곡선을 구축하기 위해 추가적인 표준 프로세스를 반복합니다. 최대 일곱 기준은 사용할 수 있습니다. 소프트웨어는 5 각 표준에 대한 복제를 저장할 수 있도록 설계되었습니다. 표준의 복제는 샘플 농도가 자동으로 결정되는에서 표준 곡선을 생성하기 위해 평균입니다. </li><li> 표준 곡선이 완료되면, &quot;샘플&quot;탭을 선택하고 각각의 샘플 ID 정보를 입력하고 각각의 미지의 샘플을 측정합니다. </li></ol>

<ol>
	<li>Wilfinger, W. W., Mackey, K., Chomczynski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+and+Ionic+Strength+on+the+Spectrophotometric+Assessment+of+Nucleic+Acid+Purity.">Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity.</a> BioTechniques. 22, 474-481 (1997).</li><li>Voolstra, C., Jungnickel, A., Borrmann, L., Kirchner, R., Huber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+Quantification+of+Nucleic+Acids:+LabelGuard+enables+photometric+quantification+of+submicroliter+samples+using+a+standard+photometer.">Spectrophotometric Quantification of Nucleic Acids: LabelGuard enables photometric quantification of submicroliter samples using a standard photometer.</a> Implen Applications Note. , (2006).</li><li>Jr, I. n. g. l. e., D, J., Crouch, S. 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> Spectrochemical analysis. , XV-590 (1988).</li><li>Van Lancker, M., Gheyssens, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+four+frequently+used+assays+for+quantitative+determination+of+DNA.">A comparison of four frequently used assays for quantitative determination of DNA.</a> Anal. Lett. 19, 615-623 (1986).</li></ol>

필립 데자르뎅와 데브라 콘클린이 문서에서 사용되는 악기를 생산 써모 과학 NanoDrop 제품, 병원에서 근무하고 있습니다.

여기에 제시 quantitation 프로토콜은 가장 널리 인정 microvolume 시스템을 기반으로합니다. 이러한 시스템은 큰 샘플 볼륨 억제 장치의 사용에 의존하는 전통적인 핵산 quantitation 방법론에 대한 실질적인 대안을 제공합니다. NanoDrop microvolume 기술은 액체 칼럼을 형성 측정되는 시료의 표면 장력 특성에 의존 샘플 보존 시스템을 사용합니다. 이것은 예제는 적절한 열 형성을위한 상위 및 하위 광학 측정 표면과 접촉을하게되는 중요합니다. 측정 결과가 부정확하거나 재현할 수없는 것 어떤 시점에서면, 그것은 대부분 샘플 이질이나 액체 열 파손의 결과입니다. 세제 및 이소 프로필 알코올은 그들이 클수록 측정 표면을 uncondition 수 있으므로 요원을 청소하지 않는 것이 좋습니다. 무조건 받침대는 샘플 비말의 평평화 그 결과, 받침대의 소수성 표면 특성이 손상되었을 경우에 발생합니다. 무조건 받침대는 측정하는 동안 액체 칼럼의 파손으로 이어질 수 있습니다. <img src="/files/ftp_upload/2565/2565fig3.jpg" alt="그림 3" /> 그림 3. 무조건 표면은 물의 비말의 평평화이 특징입니다. Uncondtioned 페데 스탈의 표면은 NanoDrop 받침대 Reconditioning 컴파 운드 (PR - 1, 써모 과학 NanoDrop 제품)를 사용 reconditioned 수 있습니다. reconditioning 화합물의 얇은 레이어 ~ 30 초 동안 건조 허용 상단 및 하단 받침대 표면에 적용되는 다음, 깨끗하고, 건조, 보푸라기가없는 실험실을 사용하여 닦아에서 문질러서. 성공적으로 reconditioned 상태는 바닥 받침대 표면에 적용할 때 구슬 세공 물 비말에 의해 특징입니다. <img src="/files/ftp_upload/2565/2565fig4.jpg" alt="그림 4" /> 그림 4. reconditioned 표면은 물의 비말 최대 구슬 장식이 특징입니다. Microvolume의 quantitation 시스템은 크게 샘플 소비를 줄이고보다 전통적인 부량 시스템에 비해 급격하게 농도 범위를 향상시킬 수 있습니다. microvolume의 quantitation는 종종 제한된 세포 바늘 biopsies 및 레이저 캡처 microdissection 같은 대량의 효율성과 편의의 사용이 방법론은 만들었습니다 그것 널리 인정하는 대신 전통적인 핵산 quantitation 방법에 관련된 상황에 대한 사용 기술되어 있지만도 샘플 풍부한 경우.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>NanoDrop 2000c</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>ND-2000C</td>
<td>&nbsp;</td>
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<td>NanoDrop 3300</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>ND-3300</td>
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nanodrop microvolume quantitation of nucleic acids

Biomolecular의 assays은 지속적으로 자주 microvolume의 quantitation를 수행할 수있는 사람들을 위해 핵산 quantitation에 대한 기존 쿠베트 기반 악기의 사용을 precluding, 재료의 점차 작은 금액을 사용하는 것이 개발되고있다. NanoDrop microvolume 샘플 유지 시스템 (써모 사이 언티픽 NanoDrop 제품) 등 cuvettes이나 모세 혈관과 같은 전통적인 억제 장치의 독립적인 시료의 미세한을 캡처하고 유지하기 위해 광섬유 기술과 자연의 표면 장력 특성을 결합하여 기능을 수행합니다. 또한, 시스템은 본질적으로 dilutions을 수행하기 위해 필요가 없습니다. 짧은 경로 길이, 핵산 농도 측정의 광범위한 결과를, 고용 spectroscopic 분석에 필요한 샘플의 볼륨을 줄이면 또한, 많은 분자 흐름 전반에 걸쳐 추가적인 품질 관리 단계의 포함을 용이 효율성을 증가하고 궁극적으로 하류 결과에 큰 자신감을 선도. 제한된 질량 높은 감도 형광 분석을위한 필요는 또한 최근의 실험 발전과 함께 등장했습니다. 같은 microvolume 샘플 보존 기술을 사용하여 형광 측정은 형광 assays 볼륨 요구 사항이 대폭 줄일 수 있도록 재료의 2 μL을 수행할 수 있습니다. 10 μL 이하의 이러한 microreactions는 전용 microvolume의 fluorospectrometer를 사용하여 지금은 가능합니다. 두 microvolume 핵산 quantitation 프로토콜은 전통적인 쿠베트 기반 프로토콜에 실질적인 대안으로 통합 샘플 보존 시스템을 사용하는 것이 증명됩니다. 첫째, microvolume 분광 광도계를 사용하여 직접 A260 흡광도 방법을 설명합니다. 이 microvolume의 fluorospectrometer로 감소 볼륨 형광 반응을 사용할 수있는 형광 기반의 방법을 시범옵니다. 이 소설 기법은 1에서 PG / μL 시료의 최소한의 소비와 15,000 NG / μL까지 핵산 농도의 평가를 가능하게합니다.

Watch this Scientific Journal Video about NanoDrop Microvolume Quantitation of Nucleic Acids at JoVE.com

NanoDrop Microvolume Quantitation of Nucleic Acids

전통적인 핵산 quantitation 방법론을 실천하고 효율적인 대안으로 NanoDrop microvolume 시스템의 사용은 두 microvolume 핵산 quantitation 프로토콜의 시범을 통해 설명합니다.

핵산의 NanoDrop Microvolume의 Quantitation

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

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nanodrop-microvolume-quantitation-of-nucleic-acids

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비디오: NanoDrop Microvolume Quantitation of Nucleic Acids

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

NanoDrop 2000c 분광을 사용 Microvolume 단백질 농도 결정

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

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생물학

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

조직 샘플에서 γH2AX Foci의 Quantitation

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

마이크로 볼륨 바이오 사양 나노 분광 광도계를 사용하여 핵산 및 단백질 농도 결정

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

전기 - 무료, 선형 핵산 및 단백질 분리

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

3DNA과 핵산의 구조를 분석하고 구축

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

에있는 고도 불포화 지방산의식이 보충제<em&gt; 예쁜 꼬마 선충</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Methyltransferases과 공동 인자 유사체와 핵산과 단백질의 순서 특정 레이블

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

높은 처리량 시퀀싱에 의한 마이크로 RNA 정량을위한 작은 RNA 분자의 고효율 내고

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

내피 세포 접착 정량<em&gt; 체외</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

디지털 핵산 정량 분석​​ 실험의 단순 대량 판독

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Methods Article

핵산의 NanoDrop Microvolume의 Quantitation