데이터의 그래픽 표현에 앤드류 랭의 지원에 감사드립니다. 실험 작업을 수행하는 장비 및 자료를 제공 메틀러 - 톨레도 감사합니다.

소개 Ugi 반응 빠르게 화합물의 다양한 라이브러리 (1-3)을 만들 수있는 편리한 방법으로 증명했다. 그것은 아민의 반응, 알데히드, 카르복실산 및 실온에서 메탄올에서 일반적으로 isonitrile을 포함한다. Ugi 반응은 종종 pharmacologically 활성 분자의 합성에 도구로 사용되고 있습니다 우리는 신속하게 새로운 안티 - 말라리아 대리인 (4)에 대한 검색 화합물을 액세스하는 그것을 이용해 왔습니다. 그것은 Ugi 제품은 때때로 반응 혼합물 (1.5)에서 순수한 형태로 침전되는 관찰되었습니다. 이것은 반응은 다음과 같은 쉽게 크로마 토그래피와 같은 비용이 많이 드는 정화 절차를 필요없이 확장할 수 있으므로 매우 운이 결과입니다. 그것은 더 이상 치료하지 않고 반응 혼합물을 여과에서 직접 얻은 Ugi 제품 수율을 최적화하기 위해 가장 도움이 될 것입니다. 이를 위해 우리는 여과 튜브를 갖춘 48 슬롯 메틀러 - 톨레도 MiniBlock (6) 활용. 메틀러 - 톨레도 MiniMapper (7) 자동 액체 핸들러는 시약과 용매를 제공하기 위해 사용되었습니다. 관심의 매개 변수는 농도, 용매 조성과 시약의 일부 초과되었습니다. 실험 추가 용매, furfurylamine (메탄올에 2M), benzaldehyde (메탄올에 2M), BOC - 글리신 (메탄올에 2M) : MiniMapper 자동 액체 처리기는 48 위치 MiniBlock에서 빈 필터 튜브에 다음 순서대로 액체를 제공하는 프로그램되었습니다 그리고 T - butylisocyanide (메탄올에 2M). 기본 추가 볼륨은 100 microliters되었습니다. 시약이 초과에 추가하다고 지적하는 경우, 120 microliters가 배달되었습니다. MiniBlock 그런 다음 집에 진공 청소기를 사용하여 필터링되기 전에 16시간 위해 쉐이크에 배치되었다. 두 세척이 해제 필터링하기 전에 흔들어 15 분 뒤에 각각의 튜브에 메탄올 (1 ML)을 추가하여 수행됩니다. 튜브는 다음 최소한 30 분 dessicator 높은 진공 하에서 건조되었다. 항복은 필터 튜브의 무게가 증가으로부터 계산되었다. 순도는 각 용매 시스템 및 농도로부터 시료에 대한 H NMR에 의해 평가되었다. 반응은 삼배하다 (80-10)에서 실행되었고 평균 수확량은 표 1에보고됩니다. Ugi 제품의 특성 <a href="http://www.chemspider.com/Chemical-Structure.21106059.html">tert - 부틸 (2 - {[2 - (tert - butylamino) - 2 - 옥소 - 1 - 페닐] (퓨란 - 2 - ylmethyl) 아미노} - 2 - oxoethyl) 카르 바 메이트</a> : 백색 고체, MP (11) 202-204 C, 1H NMR (12, <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/Exp203A11-HNMR.html">스펙트럼</a> ) (500MHz, δppm, CDCl3) 1.33 (S, 9H), 1.45 (S, 9H), 4.21 (m, 2 시간), 4.49 (D, J = 18Hz, 1H), 4.50 ( D, J = 18Hz, 1H), 5.47 (S, 1H), 5.60 (S, 1H), 5.62 (S, 1H), 5.89 (S, 1H), 6.10 (S, 1H), 7.19 (S, 1H) , 페닐 7.21-7.37 (m, 5H), 13C NMR (13, <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/Exp02060822080933CNMR.html">스펙트럼</a> ) (500MHz, δppm, CDCl3) 28.3, 28.6, 42.3, 42.8, 51.7, 62.9, 79.5, 107.7, 110.4, 128.5, 128.7, 129.6, 134.7 , 141.9, 149.8, 155.7, 168.4 170.2, IR (14, <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/ugiproductpaper03IR.html">스펙트럼</a> ) (Ʋmax cm - 1 ATR) : 1645,1673,1699, 3331, FAB - 인적 자원 관리 시스템 (15, <a href="http://showme.physics.drexel.edu/mirza/Exp099Chighres.JPG">스펙트럼</a> ) (C24H33N3O5에 대해 계산된 M / Z 444.2498이 [M + H] 444.2517을 획득하였습니다.) 데이터 분석 석출물의 항복 데이터는 단일 요소 또는 두 개의 팩터 (복제)를 Microsoft Excel에서 사용 가능한 분산 (ANOVA) 분석 도구를 사용하여 분석했다. (16) ANOVA 분석에서 통계적으로 의미있는 것으로 판명 그 변수에, 피셔의 최하위 차이 (LSD)를 테스트 Ugi의 석출물의 수확량에 큰 차이가 (17)을 전시 구체적인 실험 설정을 결정하는 데 사용되었다. 모든 중요한 검사는 95 % 신뢰 수준에서 수행되었다. 결과 및 토론 그림 1은 Ugi의 석출물의 수확량에 대한 용매 조성물의 효과를 요약한 것입니다. 단일 계수 ANOVA는 0.2M의 시약 농도에서 공부 네 가지 용매에 대한 평균 수확량에 큰 차이를 나타냅니다. 피셔의 LSD 시험은 메탄올과 에탄올에서 얻어진 침전물의 수율은 95 % 신뢰 수준 (그림 용제의 이름을 통해 바에 의해 의미)에서 통계적으로 다르​​지 않다는 것을 나타냅니다; Ugi의 석출물의 수율은 에탄올과 메탄올이 크게 크다 차례로 THF에서보다 훨씬 큰 acetonitrile,보다. <img src="/files/ftp_upload/old/942_2008_9_26_0/942_Figure-01.png" alt="그림 1" /> 그림 1 : Ugi의 수확량에 대한 용매 조성물의 효과의 요약이 reage에 침전물0.2M의 NT의 농도. 나열된 용매는이 농도에서 40 % 메탄올을 포함합니다. 시약 농도의 효과를 고려, 표 1의 데이터는 0.2M의 용매로 메탄올, 에탄올 및 acetonitrile을 사용하여 0.07M 갈 때 수확량의 북극 얼음층의 두께가 발견되었습니다 표시, 수확량은 0.2M에 메탄올에 비교되었으며 0.4 M의 농도. Ugi 반응은 일반적으로 0.5 - 2M 범위 (2)에서 실행됩니다. 그러나, 자동 액체 핸들러를 사용하는 경우, 그것은 따라서 약 0.5M의 최종 농도에 대한 실질적인 상한을 배치, 훨씬 더 집중 2M 이상의 시약 솔루션으로 시작하기 어려울 수 있습니다. 비선형 효과를 재생에 분명히 있기 때문에이 최적화 연구의 결과는, 0.2M 아래 Ugi 반응의 성공 또는 실패를 해석에주의하시기 바랍니다. 그림 2는 0.2M의 시약 농도에서 Ugi의 석출물의 수확량에 대한 용매 조성과 시약의 초과의 효과를 요약한 것입니다. 두 요인 ANOVA 결과가 통계적으로 의미 용매 및 시약의 과도한 효과를 나타냅니다. 또한, 두 변수 사이에 통계적으로 의미 상호 작용이 있습니다. 상호 작용의 세부 사항은 침전물의 수확량이 사용하는 용매에 의해 영향을 것을 보여주는 그림 2에서 밝혀낸 있습니다. 다시 그림에있는 요소를 통해 바의 수율은 95 % 신뢰 수준에서 통계적으로 다르​​지 않아 의미합니다. acetonitrile을 위해, 크게 향상된 결과를 초과 아민, 알데히드 또는 isonitrile로 취득하는 동안 에탄올과 메탄올이 모두 최고의 수율은 초과 imine 또는 isonitrile 함께 얻을 수 있습니다. 표 1의 결과의 논의 위에 바와 같이, 석출물의 전체적인 수확량은 용매로서 THF 낮은이며, imine만을 초과가 촉진 수율에 통계적으로 의미 향상에 연결됩니다. 네 가지 용매에 대한 향상된 수율 이러한 다양한 패턴을 통계적으로 중요한 상호 작용 효과에 이르게 무엇이며, 서로 다른 용매로​​ Ugi 제품의 다양한 상호 작용의 지표 수 있습니다. 표 1의 수치 결과는 최고의 생산량을 초과 imine 1.2 등가물, 0.2M acetonitrile / 메탄올 또한 상당한 수익률을 제공 0.2M 에탄올 / 메탄올 혼합과 0.4M 메탄올에서 얻은 보여주지만. 0.2M에서 THF / 메탄올 혼합의 아주 가난한 결과를 준 특히합니다. Ugi 반응이 성공적으로 THF (2)를 포함한 다양한 용매에서 실시되었습니다. 여기에 낮은 수율이 Ugi 반응은 침전물이 형성된 게 아니라 오직, THF의 혼합물에서 개최하지 않았다는 암시하지 않습니다. <img src="/files/ftp_upload/old/942_2008_10_13_5/942_Figure-02.png" alt="그림 2" width="335" height="325" /> 그림 2 : Ugi의 수확량에 대한 용매 조성과 시약의 초과의 효과의 요약은 0.2M의 시약의 농도에서 침전. 나열된 용매는이 농도에서 40 % 메탄올을 포함합니다. 그림 3은 용매로 메탄올을 사용하는 석출물 Ugi의 항복에 시약 농도와 시약의 초과의 효과를 요약한 것입니다. 두 요인 ANOVA 결과가 통계적으로 의미 농도 및 시약의 과도한 효과를 나타냅니다. 위에서 설명한 바와 같이, 0.2M 및 0.4M이 0.07M보다 훨씬 큰의의 시약의 농도에서 침전의 수율 (하지만 서로 다를하지 않음). 또한, 연구에서 두 변수 사이에 통계적으로 의미 상호 작용이 있습니다. 초과 시약의 ID와 석출물 수율의 향상의 패턴이 높은 시약의 농도에서 일반적으로 동일합니다. <img src="/files/ftp_upload/old/942_2008_9_26_2/942_Figure-03.png" alt="그림 3" width="384" height="255" /> 그림 3 : 농도와 Ugi의 항복에 시약의 초과의 효과의 요약 메탄올에 용제을 촉진. 전반적으로, 그림 2와 3에서 설명하는 결과는 용매 선택, 시약 농도와 초과하는 시약의 정체성 사이의 상호 작용이있을 거라는 것을 보여주지만, 전반적인 이들은 직선 용매 및 농도 효과보다 더 미묘했다. Ugi 반응 (18)를 최적화하면 다른 사람들이 유사한 결과를 발견했다. 결론 최적화 실험 1.2 EQ와 0.4 M의 메탄올로부터 66% 수 48 실행의 가장 높은 수율을 발견했습니다. imine니다. 이것은 크게 메탄올의 0.4 M 농도에서 몰에서 초기 반응에서 얻은 49 % 이상 얻을 수 있습니다. 석출물의 수율의 좋은 재현성을 긍정적으로 식별되는 미묘한 상호 작용 효과에 대한 허용이 복제 실험에서 얻은 것입니다.

<ol>
	<li>Marcaccini, S., Torroba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+Ugi+four-component+condensation.">The use of the Ugi four-component condensation.</a> Nature Protocols. 2, 632-63 (2007).</li><li>Domling, A., Ugi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicomponent+reactions+with+isocyanides+Angew.">Multicomponent reactions with isocyanides Angew.</a> Chem. Int. Eng. Ed. 39, 3168-31 (2000).</li><li>Domling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Developments+in+Isocyanide+Based+Multi-Component+Reactions+in.">Recent Developments in Isocyanide Based Multi-Component Reactions in.</a> Applied Chemistry, Chem. Rev.. 106, (2006).</li><li>Bradley, J. -. 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> http://usefulchem.blogspot.com/2008/01/we-have-anti-malarial-activity.html. , .</li><li>Sanudo, M., Marcaccini, M., Basurto, S., Torroba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+3-Hydroxy-6-oxo[1,2,4]triazin-1-yl+Alaninamides,+a+New+Class+of+Cyclic+Dipeptidyl.">Synthesis of 3-Hydroxy-6-oxo[1,2,4]triazin-1-yl Alaninamides, a New Class of Cyclic Dipeptidyl.</a> Ureas J. Org. Chem.. 71, 4578-45 (2006).</li><li>Bradley, J. -. C., Mirza, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> UsefulChem EXP201 http://usefulchem.wikispaces.com/EXP201. , .</li><li>Bradley, J. -. C., Mirza, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EXP202 http://usefulchem.wikispaces.com/EXP202. , .</li><li>Bradley, J. -. C., Mirza, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EXP203 http://usefulchem.wikispaces.com/EXP203. , .</li><li>p, m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> from compound UC-099C from EXP099 http://usefulchem.wikispaces.com/EXP099. , .</li><li>Bradley, J. -. C., NMR, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> spectrum 203A11 from UsefulChem EXP203 http://usefulchem.wikispaces.com/EXP203. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C NMR spectrum 206A from UsefulChem EXP206 http://usefulchem.wikispaces.com/EXP206. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IR spectrum from compound UC-099C from EXP099 http://usefulchem.wikispaces.com/EXP099. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FAB-MS from compound UC-099C from EXP099 http://usefulchem.wikispaces.com/EXP099. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis details available at http://usefulchem.wikispaces.com/space/showimage/EXP201-203_KO-A.xls. , .</li><li>Montgomery, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Analysis+of+Experiments.">Design and Analysis of Experiments.</a> Nature Protocols. , (2005).</li><li>Tye, H., Whittaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+Design+of+Experiments+approach+for+the+optimisation+of+a+microwave+assisted+Ugi+reaction.">Use of a Design of Experiments approach for the optimisation of a microwave assisted Ugi reaction.</a> Org. Biomol. Chem.. 2, 813-813 (2004).</li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Furfurylamine</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F20009</td>
<td>&nbsp;</td>
<tr>
<td>Benzaldehyde</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>12010</td>
<td>&nbsp;</td>
<tr>
<td>Boc-glycine</td>
<td>Reagent</td>
<td>TCI America</td>
<td>B1185</td>
<td>&nbsp;</td>
<tr>
<td>t-butylisocyanide</td>
<td>Reagent</td>
<td>TCI America</td>
<td>B1274</td>
<td>&nbsp;</td>		</tbody>
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optimization of the ugi reaction using parallel synthesis and automated liquid handling

furfurylamine, benzaldehyde, BOC - 글리신과 T - butylisocyanide의 혼합 관련된 Ugi 반응의 최적화가 설명되어 있습니다. 세중의는 농도, 용매 및 시약의 일부 초과 변화,보고 48 병렬 실험 실행됩니다. 제품의 절연은 간단한 여과 및 세척 절차에 의해 달성되었다. 얻은 가장 높은 수확량은 1.2 EQ와 0.4 M의 메탄올에서 66%되었다. imine니다. 이것은 크게 메탄올의 0.4 M 농도에서 몰에서 초기 반응에서 얻은 49 % 이상 얻을 수 있습니다. 0.07M에서 모든 용매 시스템이 저조한 수행하는 동안 0.4M 또는 0.2M의 시약의 농도로 메탄올 솔루션은 뛰어난 산출했다. THF / 메탄올 (40분의 60)는 가난 했었고 acetonitrile / 메탄올 (40분의 60)는 중간 동안 0.2M, 메탄올 및 에탄올 / 메탄올 (40분의 60) 혼합물에서 통계적으로 동등하게 잘되었다. 석출물의 수율의 좋은 재현성을 긍정적으로 식별되는 미묘한 상호 작용 효과에 대한 허용이 복제 실험에서 얻은 것입니다.

Watch this Scientific Journal Video about Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling at JoVE.com

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

Ugi 반응 빠르게 화합물의 다양한 라이브러리를 만들 수있는 편리한 방법으로 증명했다. 그것은 아민의 반응, 알데히드, 카르복실산 및 실온에서 메탄올에서 일반적으로 isonitrile을 포함한다. 이 비디오에서는, 우리는 여과 튜브와 메틀러 - 톨레도 MiniMapper 자동 액체 처리기는 시약 및 용매를 제공하기 위해 사용되었습니다 갖춘 48 슬롯 메틀러 - 톨레도 MiniBlock를 활용. 관심의 매개 변수는 농도, 용매 조성과 시약의 일부 초과되었습니다.

병렬 합성 및 자동 액체 처리를 사용하여 Ugi 반응의 최적화

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

optimization-ugi-reaction-using-parallel-synthesis-automated-liquid

Research

JoVE Journal

Biology

16.2K 조회수.  Drexel University. Ugi 반응 빠르게 화합물의 다양한 라이브러리를 만들 수있는 편리한 방법으로 증명했다. 그것은 아민의 반응, 알데히드, 카르복실산 및 실온에서 메탄올에서 일반적으로 isonitrile을 포함한다. 이 비디오에서는, 우리는 여과 튜브와 메틀러 - 톨레도 MiniMapper 자동 액체 처리기는 시약 및 용매를 제공하기 위해 사용되었습니다 갖춘 48 슬롯 메틀러 - 톨레도 MiniBlock?...

비디오: Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

작은 하트의 세미 - 자동 광학 도면 분석

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

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생물학

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

중합 효소 사슬 반응 : 기본 프로토콜 플러스 문제 해결 및 최적화 전략

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals across the landscape is of primary importance in watershed management. Traditional economics methods of finding the lowest-cost solution in the watershed context (e.g.,5,12,20) assume that off-site impacts can be accurately described as a proportion of on-site pollution generated. Such approaches are unlikely to be representative of the actual pollution process in a watershed, where the impacts of polluting sources are often determined by complex biophysical processes. The use of modern physically-based, spatially distributed hydrologic simulation models allows for a greater degree of realism in terms of process representation but requires a development of a simulation-optimization framework where the model becomes an integral part of optimization.
Evolutionary algorithms appear to be a particularly useful optimization tool, able to deal with the combinatorial nature of a watershed simulation-optimization problem and allowing the use of the full water quality model. Evolutionary algorithms treat a particular spatial allocation of conservation practices in a watershed as a candidate solution and utilize sets (populations) of candidate solutions iteratively applying stochastic operators of selection, recombination, and mutation to find improvements with respect to the optimization objectives. The optimization objectives in this case are to minimize nonpoint-source pollution in the watershed, simultaneously minimizing the cost of conservation practices. A recent and expanding set of research is attempting to use similar methods and integrates water quality models with broadly defined evolutionary optimization methods3,4,9,10,13-15,17-19,22,23,25. In this application, we demonstrate a program which follows Rabotyagov et al.'s approach and integrates a modern and commonly used SWAT water quality model7 with a multiobjective evolutionary algorithm SPEA226, and user-specified set of conservation practices and their costs to search for the complete tradeoff frontiers between costs of conservation practices and user-specified water quality objectives. The frontiers quantify the tradeoffs faced by the watershed managers by presenting the full range of costs associated with various water quality improvement goals. The program allows for a selection of watershed configurations achieving specified water quality improvement goals and a production of maps of optimized placement of conservation practices.

This work demonstrates an integration of a water quality model with an optimization component utilizing evolutionary algorithms to solve for optimal (lowest-cost) placement of agricultural conservation practices for a specified set of water quality improvement objectives. The solutions are generated using a multi-objective approach, allowing for explicit quantification of tradeoffs.

SWAT 모델을 사용하여 농업 보호 관행의 공간 Multiobjective 최적화 및 진화 알고리즘

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

에어 리퀴드 인터페이스에서 인간의 비강 상피 세포의 배양

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

ImageJ에 플러그인을 사용하여 셀 카운팅 절차의 자동화 된 정량 및 분석

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

절차 및 자동화 된 모 세관 전기 이동 기반 Immunoassay 방법에 대 한 중요 한 최적화 전략

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

형광, 섭취 곤충에 액체 이해 능력을 결정 하기 위한 자성 나노 입자

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

자동 이미징 시스템을 사용한 iPSC의 합류점 측정.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.