L&#39;assistenza di Andrew Lang nella rappresentazione grafica dei dati è apprezzato. Grazie alla Mettler-Toledo per la fornitura di attrezzature e materiali per svolgere il lavoro sperimentale.

Introduzione La reazione di Ugi ha dimostrato di essere un modo conveniente per creare velocemente librerie di composti diversi (1-3). Coinvolge la reazione di un ammina, un&#39;aldeide, un acido carbossilico e un isonitrile tipicamente in metanolo a temperatura ambiente. La reazione di Ugi è stato spesso utilizzato come strumento nella sintesi di molecole farmacologicamente attive e l&#39;abbiamo sfruttata per accedere rapidamente composti nella ricerca di nuovi agenti anti-malariche (4). E &#39;stato osservato che i prodotti Ugi a volte precipitare in forma pura dalla miscela di reazione (1,5). Si tratta di un risultato molto fortunato, perché la reazione può quindi essere facilmente scalati senza richiedere costose procedure di purificazione, come la cromatografia. Sarebbe più vantaggioso per ottimizzare la resa del prodotto Ugi quanto ottenuto direttamente dal filtraggio della miscela di reazione senza ulteriori trattamenti. A questo fine abbiamo utilizzato un 48 slot Mettler-Toledo MINIBLOCK (6) dotati di tubi di filtrazione. Un Mettler-Toledo MiniMapper (7) gestore automatico liquido è stato utilizzato per fornire i reagenti e solventi. I parametri di interesse sono stati la concentrazione, la composizione del solvente e l&#39;eccesso di alcuni dei reagenti. Sperimentale Il gestore automatico MiniMapper liquido è stato programmato per fornire liquidi nella seguente sequenza di svuotare i tubi del filtro in 48 posizione MINIBLOCK: ulteriori solventi, furfurylamine (2M in metanolo), benzaldeide (2M in metanolo), Boc-glicina (2M in metanolo) e t-butylisocyanide (2M in metanolo). Il volume aggiunta di default era 100 microlitri. Se un reagente è notato come aggiunto in eccesso, 120 microlitri sono stati consegnati. Il MINIBLOCK è stato poi posto su un agitatore per 16 ore prima di essere filtrato usando il vuoto casa. Due lavaggi vengono eseguiti con l&#39;aggiunta di metanolo (1 ml) a ciascuna provetta seguita da 15 minuti agitando prima di filtrare fuori. I tubi sono poi stati essiccati sotto alto vuoto in essiccatore per almeno 30 min. Il rendimento è stato calcolato l&#39;aumento di peso dei tubi del filtro. La purezza è stata valutata da H NMR di un campione di ciascun sistema solvente e la concentrazione. Le reazioni sono state eseguite in triplice copia (8-10) e le rese medie sono riportati nella tabella 1. Caratterizzazione del prodotto Ugi <a href="http://www.chemspider.com/Chemical-Structure.21106059.html">terz-butile (2 - {[2 - (terz-butilammino)-2-oxo-1-feniletil] (furano-2-ilmetil) ammino}-2-oxoethyl) carbammato</a> : solido bianco; mp (11) 202-204 C; 1H NMR (12, <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/Exp203A11-HNMR.html">spettro</a> ) (500MHz, 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) , fenile 7,21-7,37 (m, 5H), 13C NMR (13, <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/Exp02060822080933CNMR.html">spettro</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">spettro</a> ) (Ʋmax cm-1 ATR): 1645,1673,1699, 3331; FAB-HRMS (15, <a href="http://showme.physics.drexel.edu/mirza/Exp099Chighres.JPG">spettro</a> ) (calcolato per C24H33N3O5 m / z 444,2498 [M H +], ottenuto 444,2517). Analisi dei dati I dati di precipitazione di rendimento sono stati analizzati utilizzando il singolo fattore o due fattori (con repliche) analisi della varianza (ANOVA) gli strumenti disponibili in Microsoft Excel. (16) Per queste variabili risultate statisticamente significative per l&#39;analisi ANOVA, piccola differenza significativa di Fisher (LSD) test è stato utilizzato per determinare le impostazioni sperimentali specifiche che presentano differenze significative nel rendimento del precipitato Ugi (17). Tutti i test di significatività sono state eseguite a livello di confidenza del 95%. Risultati e discussione La Figura 1 riassume l&#39;effetto della composizione del solvente sul rendimento del precipitato Ugi. Una ANOVA singolo fattore indica una differenza significativa nella resa media per i quattro solventi studiato ad una concentrazione di 0,2 M reagente. Test di LSD di Fisher indica che le rese precipitato ottenuto in metanolo ed etanolo non sono statisticamente differenti al livello di confidenza del 95% (indicato dal bar sopra i nomi di solventi in figura), la resa del precipitato Ugi è significativamente maggiore in etanolo e metanolo che in acetonitrile, che a sua volta è significativamente maggiore di quella in THF. <img src="/files/ftp_upload/old/942_2008_9_26_0/942_Figure-01.png" alt="Figura 1" /> Figura 1: Sintesi degli effetti della composizione del solvente sul rendimento di Ugi precipitare in un Reageconcentrazione di 0,2 M nt. I solventi elencati comprendono 40% di metanolo a questa concentrazione. Considerando l&#39;effetto della concentrazione dei reagenti, i dati nella tabella 1 mostra un precipitoso calo di rendimento è stato trovato quando si passa da 0,2 M a 0.07M con metanolo, etanolo e acetonitrile come solvente, i rendimenti erano paragonabili in metanolo al 0,2 M e 0,4 M concentrazioni. Si noti che le reazioni Ugi sono generalmente eseguito in 0,5-2M gamma (2). Tuttavia, quando si utilizza un gestore automatico liquido, può essere difficile partire con soluzioni reagenti molto più concentrato di 2M, così ponendo un limite pratico superiore alla concentrazione finale di 0,5 M about. I risultati di questo studio di ottimizzazione suggerisce cautela nell&#39;interpretare il successo o il fallimento di reazioni di Ugi sotto 0,2 M, dato che effetti non lineari sono apparentemente in gioco. La Figura 2 riassume l&#39;effetto della composizione del solvente e reagente in eccesso sul rendimento del precipitato Ugi ad una concentrazione di 0,2 M reagente. I due fattori risultati ANOVA indicare un solvente e l&#39;eccesso statisticamente significativo effetto dei reagenti. Inoltre, vi è una interazione statisticamente significativa tra le due variabili. Dettagli delle interazioni sono scoperti in figura 2 che mostra che il rendimento del precipitato è influenzato dal solvente utilizzato. Anche in questo caso di notare che le barre sui fattori nella figura indicano le rese non sono statisticamente differenti al livello di confidenza del 95%. Per entrambi l&#39;etanolo e il metanolo i prodotti migliori sono ottenuti con l&#39;immina isonitrile o in eccesso, mentre per acetonitrile, ha migliorato significativamente i risultati si ottengono con l&#39;aldeide ammina, o isonitrile in eccesso. Come osservato in precedenza nella discussione dei risultati in tabella 1, il rendimento complessivo del precipitato è bassa per THF come solvente, e solo un eccesso di immina porta ad un miglioramento statisticamente significativo della resa precipitato. Questi diversi modelli di rendimento migliore per i quattro solventi è ciò che porta a l&#39;effetto di interazione statisticamente significativa, e può essere un indicatore di interazione differenti del prodotto Ugi con i solventi diversi. Anche se i risultati numerici nella tabella 1 mostrano il miglior rendimento è stato ottenuto in metanolo 0.4M con 1,2 equivalenti di immina eccesso, 0,2 M acetonitrile / metanolo ed etanolo 0,2 M / miscele metanolo fornito anche i rendimenti significativi. Si noti soprattutto che tutti i THF / metanolo miscele a 0,2 M ha dato risultati molto poveri. La reazione di Ugi è stato effettuato con successo in una varietà di solventi, compresi THF (2). Si noti che la bassa resa qui non implicano che la reazione di Ugi non ha avuto luogo a miscele THF, ma solo che un precipitato non si è formata. <img src="/files/ftp_upload/old/942_2008_10_13_5/942_Figure-02.png" alt="Figura 2" width="335" height="325" /> Figura 2: Sintesi degli effetti della composizione del solvente e reagente in eccesso sul rendimento di Ugi precipitato a una concentrazione di 0,2 M reagente. I solventi elencati comprendono 40% di metanolo a questa concentrazione. La Figura 3 riassume l&#39;effetto della concentrazione del reagente e reagente in eccesso sul rendimento di Ugi precipitare utilizzando metanolo come solvente. I due fattori risultati ANOVA indicano una concentrazione e l&#39;eccesso statisticamente significativo effetto dei reagenti. Come descritto sopra, il rendimento del precipitato a concentrazioni di reagenti e di 0,2 M 0.4M sono significativamente maggiori rispetto a 0.07M (ma non differiscono l&#39;uno dall&#39;altro). Inoltre, vi è una interazione statisticamente significativa tra le due variabili oggetto di studio. Si noti che il modello del miglioramento del rendimento precipitato con l&#39;identità del reagente in eccesso è generalmente la stessa alle concentrazioni dei reagenti più 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: Sintesi degli effetti della concentrazione e del reagente in eccesso sul rendimento di Ugi precipitati in metanolo solvente. Nel complesso, i risultati descritti nelle figure 2 e 3 dimostrano che vi erano le interazioni tra il solvente scelta, la concentrazione del reagente e l&#39;identità del reagente in eccesso, ma nel complesso questi erano più sottile degli effetti direttamente solvente e la concentrazione. Altri hanno trovato risultati simili per ottimizzare una reazione di Ugi (18). Conclusione L&#39;esperimento di ottimizzazione ha riscontrato il maggior rendimento delle 48 corre da 66% da 0,4 metanolo M con 1,2 eq. di immina. Questo è significativamente al di sopra del rendimento del 49% ottenuto dalla reazione iniziale in concentrazione equimolare a 0,4 M in metanolo. Buona riproducibilità delle rese precipitazione è stata ottenuta in questi esperimenti replicare, consentendo effetti di interazione sottile per essere identificato.

<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>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Furfurylamine</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F20009</td>
<td>&nbsp;</td>
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<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>
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optimization of the ugi reaction using parallel synthesis and automated liquid handling

L&#39;ottimizzazione di una reazione di Ugi che coinvolge la miscelazione di furfurylamine, benzaldeide, Boc-glicina e t-butylisocyanide è descritto. Triplice copia corre di 48 esperimenti in parallelo sono riportati, variando la concentrazione, solvente e l&#39;eccesso di alcuni dei reagenti. L&#39;isolamento del prodotto è stato ottenuto da una semplice filtrazione e procedura di lavaggio. La più alta resa ottenuta è stata del 66% da 0,4 metanolo M con 1,2 eq. di immina. Questo è significativamente al di sopra del rendimento del 49% ottenuto dalla reazione iniziale in concentrazione equimolare a 0,4 M in metanolo. Soluzioni di metanolo con concentrazioni dei reagenti di 0.4M o 0,2 M ha dato rendimenti superiori, mentre tutti i sistemi di solvente a 0.07M eseguita male. A 0,2 M, metanolo ed etanolo / metanolo (60/40) miscele erano statisticamente altrettanto bene, mentre il THF / metanolo (60/40) era povera e acetonitrile / metanolo (60/40) è stata intermedi. Buona riproducibilità delle rese precipitazione è stata ottenuta in questi esperimenti replicare, consentendo effetti di interazione sottile per essere identificato.

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 reazione di Ugi ha dimostrato di essere un modo conveniente per creare velocemente librerie di composti diversi. Coinvolge la reazione di un ammina, un&#39;aldeide, un acido carbossilico e un isonitrile tipicamente in metanolo a temperatura ambiente. In questo video, utilizziamo un 48-slot per Mettler-Toledo MINIBLOCK dotato di tubi di filtraggio e di un Mettler-Toledo liquid handler MiniMapper automatizzato è stato utilizzato per fornire i reagenti e solventi. I parametri di interesse sono stati la concentrazione, la composizione del solvente e l&#39;eccesso di alcuni dei reagenti.

Ottimizzazione della reazione Ugi Con Sintesi parallela e automatica Gestione liquidi

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 Views.  Drexel University. La reazione di Ugi ha dimostrato di essere un modo conveniente per creare velocemente librerie di composti diversi. Coinvolge la reazione di un ammina, un'aldeide, un acido carbossilico e un isonitrile tipicamente in metanolo a temperatura ambiente. In questo video, utilizziamo un 48-slot per Mettler-Toledo MINIBLOCK dotato di tubi di filtraggio e di un Mettler-Toledo liquid handler MiniMapper automatizzato è stato utilizzato per fornire i reagenti e solventi. I para...

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.

Semiautomatici Analisi Heartbeat ottica di piccoli cuori

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

Ricerca

Biologia

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.

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

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.

Polymerase Chain Reaction: protocollo Basic Plus Strategies Risoluzione dei problemi e ottimizzazione

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.

Spaziale Ottimizzazione multiobiettivo di pratiche di conservazione agricole utilizzando un modello SWAT e 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.

Coltura di cellule umane nasale epiteliali presso l&#39;Air Interface Liquid

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.

Automated quantificazione e analisi di procedure di conteggio delle cellule Uso ImageJ Plugin

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.

Procedure e strategie di ottimizzazione chiave per un metodo automatizzato Immunoassay basati su elettroforetica capillare

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.

L'ingestione di fluorescente, nanoparticelle magnetiche per determinare la capacità di assorbimento di liquido negli insetti

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.

Misurazione della confluenza di iPSC utilizzando un sistema di imaging automatizzato.

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.