Asistencia de Andrew Lang en la representación gráfica de los datos se aprecia. Gracias a Mettler-Toledo para la provisión de equipos y materiales para llevar a cabo el trabajo experimental.

Introducción La reacción de Ugi ha demostrado ser una manera conveniente de crear rápidamente diversas bibliotecas de compuestos (1-3). Se trata de la reacción de una amina, aldehído, un ácido carboxílico y un isonitrilo normalmente en metanol a temperatura ambiente. La reacción de Ugi ha utilizado a menudo como una herramienta en la síntesis de moléculas farmacológicamente activas, y lo hemos aprovechado para acceder rápidamente a los compuestos en la búsqueda de nuevos agentes anti-malaria (4). Se ha observado que los productos Ugi a veces se precipitan en forma pura de la mezcla de reacción (1,5). Este es un resultado muy afortunado ya que la reacción puede ser fácilmente ampliado sin necesidad de costosos procesos de purificación, como la cromatografía. Sería más beneficioso para optimizar el rendimiento del producto Ugi como se obtiene directamente de filtrar la mezcla de reacción sin tratamiento adicional. Con este fin se utilizó un 48 ranura Mettler-Toledo MiniBlock (6) equipados con tubos de filtración. A Mettler-Toledo MiniMapper (7) manejador de líquidos automatizados se utilizan para entregar los reactivos y disolventes. Los parámetros de interés fueron la concentración, la composición del disolvente y el exceso de algunos de los reactivos. Experimental El manejador de MiniMapper líquido automatizado fue programado para entregar líquidos en el siguiente orden de vaciar los tubos de filtro en una MiniBlock de 48 posiciones: disolvente adicional, furfurylamine (2M en metanol), benzaldehído (2 M en metanol), boc-glicina (2 M en metanol) y t-butylisocyanide (2M en metanol). Además el volumen por defecto es 100 microlitros. Si un reactivo se observa como se añade en exceso, 120 microlitros fueron entregados. El MiniBlock fue colocado en un agitador durante 16 horas antes de ser filtrado con vacío de la casa. Dos lavados se llevan a cabo mediante la adición de metanol (1 mL) a cada tubo, seguido de 15 min agitando antes de filtrado. Los tubos se secó en alto vacío en un desecador durante al menos 30 min. El rendimiento se calcula a partir del aumento en el peso de los tubos del filtro. La pureza se evaluó mediante RMN de una muestra de cada sistema de disolvente y la concentración. Las reacciones se realizaron por triplicado (8-10) y los rendimientos promedio se reportan en la Tabla 1. La caracterización del producto Ugi <a href="http://www.chemspider.com/Chemical-Structure.21106059.html">terc-butilo (2 - {[2 - (terc-butilamino)-2-oxo-1-fenil] (furan-2-ilmetil) amino}-2-oxoetil) carbamato</a> : sólido blanco, punto de fusión (11) desde 202 hasta 204 C; 1H RMN (12, <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/Exp203A11-HNMR.html">espectro</a> ) (500 MHz, δppm, CDCl3) 1,33 (s, 9H), 1,45 (s, 9H), 4,21 (m, 2H), 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) , fenilo 7,21 a 7,37 (m, 5H); 13C RMN (13, <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/Exp02060822080933CNMR.html">espectro</a> ) (500 MHz, 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">espectro</a> ) (Ʋmax cm-1 ATR): 1645,1673,1699, 3331, FAB-HRMS (15, <a href="http://showme.physics.drexel.edu/mirza/Exp099Chighres.JPG">espectro</a> ) (calculado para C24H33N3O5 m / z 444,2498 [M + H], obtenido 444,2517). Análisis de Datos Los datos de rendimiento precipitado se analizó mediante el factor individual o de dos factores (con repeticiones), análisis de varianza (ANOVA) las herramientas disponibles en Microsoft Excel. (16) Para aquellas variables que son estadísticamente significativas en el análisis de ANOVA, la mínima diferencia significativa de Fisher (LSD) se utilizó la prueba para determinar los parámetros experimentales específicas presentan diferencias significativas en el rendimiento del precipitado Ugi (17). Todas las pruebas de significación se realizaron en el nivel de confianza del 95%. Resultados y Discusión La Figura 1 resume el efecto de la composición de los disolventes en el rendimiento del precipitado Ugi. Un ANOVA de un solo factor indica una diferencia significativa en el rendimiento promedio para los cuatro solventes estudió en una concentración de reactivo de 0,2. Prueba de Fisher LSD indica que los rendimientos precipitado obtenido en metanol y etanol no son estadísticamente diferentes al nivel de confianza del 95% (representado por la barra sobre los nombres de los disolventes en la figura), el rendimiento de precipitado Ugi es significativamente mayor en etanol y metanol que en acetonitrilo, que a su vez es significativamente mayor que en THF. <img src="/files/ftp_upload/old/942_2008_9_26_0/942_Figure-01.png" alt="Figura 1" /> Figura 1: Resumen del efecto de la composición de los disolventes en el rendimiento de Ugi precipitado a un Reagent concentración de 0,2 M. Los disolventes indicados incluyen 40% de metanol a esta concentración. Teniendo en cuenta el efecto de la concentración de los reactivos, los datos del Cuadro 1 muestran una precipitada caída en el rendimiento fue encontrado al pasar de 0,2 a 0.07M con metanol, etanol y acetonitrilo como disolvente, los rendimientos fueron similares en metanol en el 0,2 y el 0,4 M concentraciones. Tenga en cuenta que las reacciones son generalmente Ugi ejecutar en el rango de 0.5-2M (2). Sin embargo, cuando se utiliza un controlador automático de líquido, puede ser difícil para empezar con las soluciones de reactivo mucho más concentrada que 2M, lo que coloca un límite superior práctico en la concentración final en alrededor de 0,5. Los resultados de este estudio de optimización sugieren cautela en la interpretación del éxito o el fracaso de las reacciones por debajo de 0,2 M Ugi, ya que los efectos no lineales son al parecer en el juego. La figura 2 resume el efecto de la composición del disolvente y el exceso de reactivos en el rendimiento de precipitado Ugi a una concentración de 0,2 M de reactivo. Los resultados de dos factores ANOVA indican un solvente y el efecto estadísticamente significativo exceso de reactivo. Además, existe una interacción estadísticamente significativa entre ambas variables. Los detalles de la interacción que se descubran en la figura 2, que muestra que el rendimiento de precipitado se ve afectada por el disolvente utilizado. Una vez más en cuenta que las barras sobre los factores de la figura indican los rendimientos no son estadísticamente diferentes al nivel de confianza del 95%. Para el etanol y el metanol los mejores rendimientos se obtienen con la imina o isonitrilo en exceso, mientras que para el acetonitrilo, mejoró los resultados significativamente se obtienen con la amina, aldehído o isonitrilo en exceso. Como se señaló anteriormente en la discusión de los resultados en la Tabla 1, el rendimiento global de precipitado es baja para el THF como disolvente, y sólo un exceso de imina conduce a una mejoría estadísticamente significativa en el rendimiento de precipitado. Estos diferentes patrones de mejor rendimiento para los cuatro solventes es lo que lleva a efecto de interacción estadísticamente significativa, y puede ser un indicador de la interacción de los diferentes productos Ugi con los solventes diferentes. A pesar de los resultados numéricos en la Tabla 1 muestra los mejores rendimientos se obtuvieron en metanol 0,4 M con 1,2 equivalentes de imina exceso de acetonitrilo 0,2 M / metanol y 0,2 M de etanol / metanol mezclas también proporcionan rendimientos significativos. Tenga en cuenta especialmente que todas las mezclas de THF / metanol a 0,2 M dio resultados muy pobres. La reacción de Ugi se ha realizado con éxito en una variedad de disolventes, incluso el THF (2). Tenga en cuenta que los bajos rendimientos de aquí no implica que la reacción de Ugi no tuvo lugar en las mezclas de THF, sólo que el precipitado no se formó. <img src="/files/ftp_upload/old/942_2008_10_13_5/942_Figure-02.png" alt="Figura 2" width="335" height="325" /> Figura 2: Resumen del efecto de la composición del disolvente y el exceso de reactivos en el rendimiento de Ugi precipitado a una concentración de 0,2 M de reactivo. Los disolventes indicados incluyen 40% de metanol a esta concentración. Figura 3 se resume el efecto de la concentración de los reactivos y el exceso de reactivo en el rendimiento de Ugi precipitar utilizando metanol como disolvente. Los resultados de dos factores ANOVA indican una concentración estadísticamente significativa y el efecto exceso de reactivo. Como se describió anteriormente, el rendimiento de precipitado en las concentraciones de reactivos de 0,2 M y 0,4 M de son significativamente mayores que en 0.07M (pero no se diferencian unos de otros). Además, existe una interacción estadísticamente significativa entre las dos variables en estudio. Tenga en cuenta que el patrón de la mejora en el rendimiento de precipitado con la identidad del reactivo en exceso suele ser el mismo en las concentraciones de reactivos más alto. <img src="/files/ftp_upload/old/942_2008_9_26_2/942_Figure-03.png" alt="Figura 3" width="384" height="255" /> Figura 3: Resumen del efecto de la concentración y el exceso de reactivos en el rendimiento de Ugi precipitado en metanol solvente. En general, los resultados descritos en las figuras 2 y 3 demuestran que hubo interacción entre la concentración de reactivo opción solvente, y la identidad del reactivo en exceso, pero en general eran más sutiles que los efectos directo solvente y la concentración. Otros han encontrado similares resultados al optimizar una reacción Ugi (18). Conclusión El experimento de optimización encuentra el mayor rendimiento de las 48 carreras que el 66% de metanol 0,4 M con 1,2 eq. de imina. Esto es muy por encima del rendimiento del 49% obtenido de la reacción inicial en la concentración equimolar de 0,4 M en metanol. Una buena reproducibilidad de los rendimientos precipitado se obtiene en estos experimentos replican, lo que permite efectos de la interacción sutil para ser identificado positivamente.

<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>
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optimization of the ugi reaction using parallel synthesis and automated liquid handling

La optimización de una reacción de Ugi participación de la mezcla de furfurylamine, benzaldehído, boc glicina-y t-butylisocyanide se describe. Por triplicado, corre de 48 experimentos paralelos se presentan, variando la concentración, disolvente y el exceso de algunos de los reactivos. El aislamiento del producto fue alcanzado por una simple filtración y el procedimiento de lavado. El mayor rendimiento obtenido fue del 66% de metanol 0,4 M con 1,2 eq. de imina. Esto es muy por encima del rendimiento del 49% obtenido de la reacción inicial en la concentración equimolar de 0,4 M en metanol. Soluciones de metanol con una concentración de reactivo de 0,4 o 0,2 M dio mejores rendimientos, mientras que todos los sistemas de disolvente a 0.07M un mal desempeño. En mezclas de 0,2 M, metanol y etanol / metanol (60/40) fueron estadísticamente igual de buenos, mientras que THF / metanol (60/40) era pobre y acetonitrilo / metanol (60/40) fue intermedio. Una buena reproducibilidad de los rendimientos precipitado se obtiene en estos experimentos replican, lo que permite efectos de la interacción sutil para ser identificado positivamente.

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

La reacción de Ugi ha demostrado ser una manera conveniente de crear rápidamente diversas bibliotecas de compuestos. Se trata de la reacción de una amina, aldehído, un ácido carboxílico y un isonitrilo normalmente en metanol a temperatura ambiente. En este video, nosotros utilizamos un 48 ranura Mettler-Toledo MiniBlock equipados con tubos de filtración y un manejador de Mettler-Toledo MiniMapper líquido automatizado fue utilizado para entregar los reactivos y disolventes. Los parámetros de interés fueron la concentración, la composición del disolvente y el exceso de algunos de los reactivos.

Optimización de la reacción de Ugi mediante la síntesis en paralelo y manejo de líquidos automatizados

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 vistas.  Drexel University. La reacción de Ugi ha demostrado ser una manera conveniente de crear rápidamente diversas bibliotecas de compuestos. Se trata de la reacción de una amina, aldehído, un ácido carboxílico y un isonitrilo normalmente en metanol a temperatura ambiente. En este video, nosotros utilizamos un 48 ranura Mettler-Toledo MiniBlock equipados con tubos de filtración y un manejador de Mettler-Toledo MiniMapper líquido automatizado fue utilizado para entregar los reactivos y dis...

Video: 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.

Semi-automatizado de análisis óptico latido de los corazones pequeños

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

Investigación

Biología

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.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

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.

Reacción en cadena: Protocolo Básico Plus Estrategias de resolución de problemas y optimización

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.

Optimización multiobjetivo espacial de las prácticas de agricultura de conservación que utilizan un modelo SWAT y un algoritmo evolutivo

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.

El cultivo de células humanas epiteliales nasales en la interfase aire líquido

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.

Automatizado de cuantificación y análisis de los procedimientos de recuento de células usando ImageJ Plugins

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.

Procedimiento y estrategias clave de la optimización para un método de inmunoensayo de basadas en electroforesis capilar automatizado

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.

La ingestión de fluorescente, nanopartículas magnéticas para determinar capacidades de absorción de fluidos en insectos

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.

Medición de la confluencia de iPSCs utilizando un sistema automatizado de imágenes.

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.