El trabajo en nuestro laboratorio con el apoyo de Career Award MCB0551798 de la Fundación Nacional de Ciencia y AREA conceder R15GM86839 de la NIGMS-NIH.

1. El crecimiento y la radiación ultravioleta. <ol><li> 200μl de un cultivo fresco durante la noche que contiene el plásmido pBR322 crecido en medio de una Davis suplementado con 0,4% de glucosa, 0,2% casaminoácidos, y 10 mg / ml timina (medio DGCthy) y 100 ug / ml ampicilina es granulado. El pellet de células se resuspende entonces en un medio de DGCthy 200μl falta de ampicilina y se utiliza para inocular 20 ml de medio DGCthy. </li><li> Los cultivos son cultivados sin selección a la ampicilina en una incubadora de agitación a 37 ° C a una OD 600 de 0,5 (~ 5 x 10 8 células / ml). Crecimiento sin ampicilina evita la selección contra intermediarios de replicación anormal o improductivos que pueden surgir en algunos mutantes. Además, si se utiliza la luz ultravioleta para inducir daño, la eliminación de la ampicilina de los medios de comunicación es necesaria, ya que absorbe fuertemente en estas longitudes de onda y los escudos de las células, la reducción de la dosis efectiva de radiación UV. </li><li> Que trabajan bajo las luces amarillas, la cultura se encuentra en un diámetro de 15cm placa de Petri en una plataforma giratoria para la agitación. Nuestras culturas se colocan a una distancia de una lámpara de 15 vatios germicida que produce una tasa de exposición de aproximadamente 1 J/m2/sec, que se mide con un fotómetro de UVC. Las culturas son irradiados con 50 J/m2 y luego se coloca inmediatamente detrás en el temblor 37 ° C incubadora para la duración del experimento. Esta dosis produce, en promedio, un ciclobutano pirimidina dímero cada 4,5 kb de ADN de cadena simple. La iluminación amarilla impide fotorreactivación-inversión de los dímeros de pirimidina ciclobutano por fotoliasa. </li></ol> 2. De aislamiento de ADN. <ol><li> En momentos en que los intermediarios de replicación se han de examinar, una alícuota de 0,75 ml de la cultura se coloca en 0,75 ml de hielo frío NET30 buffer (100 mM NaCl, 10 mM Tris, pH 8,0, 30 mM EDTA) y se coloca en hielo hasta el final de el transcurso del tiempo. Por lo general un curso de 90 minutos, con muestras examinadas a los 0, 15, 30, 45, 60 y 90 minutos. El EDTA y la temperatura fría sirven para detener efectivamente la replicación y la reparación por escisión de nucleótidos. </li><li> Cada muestra se sedimentaron, se resuspendieron en 150 ml de 1,5 mg / ml de lisozima y 0,2 mg / ml RNAseA en TE (10 mM Tris, pH 8,0, 1 mM EDTA), y lisadas a 37 ° C durante 20 min. En este tiempo, 10μl de proteinasa K (10mg/ml) y 10μl de 20% sarkosyl se añadió la incubación se permite que continúe durante 1 hora. Proteinasa K y sarkosyl ayudan a liberar fragmentos de ADN que pueden estar asociados con la membrana o las proteínas antes de la extracción con fenol se produce. Dado que el ADN en replicación activa es a menudo obligados a complejos de proteínas o de la membrana, esto ayuda a aumentar el rendimiento de la replicación de los fragmentos que han sido recuperadas. </li><li> Las muestras se extraen mediante la adición de 2 volúmenes de fenol para cada muestra y los tubos se vuelve a invertir durante 5 minutos. A continuación, dos volúmenes de cloroformo / alcohol isoamílico (24 / 1) se agregan y los tubos se vuelve a invertir de nuevo durante 5 minutos. </li><li> Las muestras se centrifugan a 14.000 rpm en una microcentrífuga durante 5 minutos y la fase superior acuosa de cada muestra se retira y se coloca en un tubo nuevo. A continuación, cuatro volúmenes de cloroformo / alcohol isoamílico (24 / 1) se añaden, los tubos se vuelve a invertir durante 5 minutos y se centrifuga de nuevo a 14.000 rpm durante 5 minutos. </li><li> La fase superior acuosa, en cada muestra se dializa durante 1 hora en manchas 100μl de cada muestra en un disco de 47 mm Whatman 0.025 micras de poro que flota en 250 ml de 0,1 X TE en un vaso. Porque las estructuras de replica con las regiones solo filamento o puntos de ramificación son más susceptibles al corte, por lo general reducir nuestras puntas de pipeta con una hoja de afeitar para hacer la boca más ancha y reducir al mínimo las fuerzas de cizallamiento. En general, las pipetas deben mantenerse al mínimo hasta que el ADN ha sido digerido con enzimas de restricción. </li><li> Cada muestra se digiere con PvuII (New England Biolabs), que alinea el plásmido justo abajo de el origen de replicación. </li><li> Antes de la carga en el gel de agarosa, el cloroformo y 100μl 20μl de colorante de carga 6X que contiene cyanol azul de bromofenol y xileno se añadió a cada muestra y se mezcla. Entonces, 40μl de la fase acuosa que contiene el colorante de carga se carga en el gel. Para minimizar los sesgos estructurales, artefactos estructurales, y cortar el ADN, el procedimiento de aislamiento no incluye ninguna medida para enriquecer a las regiones sola hebra, precipitado, o concentrar las muestras de ADN después de la lisis celular. </li></ol> 3. Gel 2D y Análisis del Sur. <ol><li> El 2-dimensional de agarosa-gel análisis se ha modificado a partir del 3. Para la 1 ª dimensión, las muestras de ADN restringido se ejecutan a través de un gel de agarosa 0,4% en TBE 1X en 1V/cm. Un litro de solución 10X TBE contiene acciones: <ul><li> 108 g de Tris Base </li><li> 55 g de ácido bórico </li><li> 40 ml de EDTA 0,5 M (pH 8,0) </li></ul></li><li> A lambda posteriores marcador de tamaño III se carga en el primer carril, las muestras se cargan en cada carril. Por lo general ejecuta la primera dIMENSIÓN ~ 15.12 hrs. Saltarse los carriles es más fácil de cortar las rutas a lanzar en la segunda dimensión. El bajo voltaje y baja por ciento en gel de agarosa sirve para separar los fragmentos de ADN sobre todo en función del tamaño. </li><li> Para la segunda dimensión, carriles de gel se cortan fuera de la primera dimensión de gel con un cuchillo de carnicero de grandes. El primer carril que contiene el marcador lambda HindIII se puede teñir con bromuro de etidio. Con el marcador lambda HindIII como guía, cortar y desechar la región del gel que se encuentra por debajo en el plásmido linealizado se espera que funcione. En estas condiciones, nos encontramos con que pBR322 linearizado encuentra ligeramente por encima de la mancha cyanol xileno. </li><li> Para lanzar la segunda dimensión, cada carril se coloca horizontalmente en la parte superior de un lanzador de gel vacías. Una solución de 1,0% de agarosa en TBE 1X se prepara y se enfría a 55 ° C. En este momento, la solución de gel se vierte en el molde para la segunda dimensión, asegurándose de cubrir completamente las rodajas de gel. Una vez que el gel se ha puesto, que se ejecuta en 6.5V/cm en una unidad de electroforesis, que permite la recirculación de la memoria intermedia. Nosotros normalmente se ejecuta la segunda dimensión ~ 5.5-7 horas. El alto voltaje y alta por ciento en gel de agarosa separa eficazmente los fragmentos de ADN en base a su forma, así como el tamaño. Formas no lineales se ejecutan más lentamente a través de la segunda dimensión. </li><li> Tras la electroforesis, el gel se enjuaga en H 2 O, se lavaron dos veces en 400 ml de ácido clorhídrico 0,25 M durante 15 minutos, se lavan con H 2 O;, y luego se lavan dos veces en 400 ml de NaOH 0,4 M durante 30 minutos. El ácido se lava sirven para nick parcialmente las moléculas de ADN en fragmentos más pequeños que la transferencia más eficiente. Este paso es necesario debido al gran tamaño y formas poco comunes de los intermediarios de replicación del ADN. </li><li> El ADN de los geles se transfiere entonces a un Hybond N + membrana de nylon 4. Usamos un sistema de baja transferencia de álcali con 0,4 M NaOH. En resumen, dos hojas de papel secante empapado en 0,4 M de NaOH se colocan en una gran pila de toallas de papel. La membrana de nylon también empapado en 0,4 M de NaOH y se coloca en la parte superior del papel secante. El gel es cuidadosamente por capas en la parte superior de este, seguido de otro pedazo de papel secante, mojada en la solución de NaOH. Por último, una larga pieza de papel secante mojado es en capas en la parte superior y sus dos extremos se colocan en una vajilla que contiene 1 L de solución de NaOH 0,4 M para servir como una mecha. Por lo general permiten la transferencia de ADN de 6-12 hrs. </li><li> La membrana de nylon, se lava 20-30 segundos en Buffer 5X SSC, y se coloca en una botella de rodillos de hibridación que contenía solución de prehibridación 10.5ml. A continuación, la membrana se incuba a 42 ° C con rotación por más de 6 horas. Prehibridación solución: <ul><li> 5,0 ml de formamida </li><li> 0,5 ml de 20% SDS </li><li> 2,5 ml de 20X SSC * </li><li> * 2.0 ml 50X Denhardt </li><li> 0,5 ml de esperma de salmón de ADN (10mg/ml) * Recetas para la CSS y de Denhardt se puede encontrar en cinco </li></ul></li><li> Durante el período de prehibridación, 1 g de plásmido pBR322 es marcado con 32P por nick de traducción de acuerdo con el protocolo suministrado por Roche con la etiqueta alfa [32 P]-dCTP. La sonda marcada radiactivamente (100μl) se desnaturaliza mediante la incubación a 98 ° C durante 10 minutos en un tubo de microcentrífuga de tornillo de la tapa y se coloca inmediatamente en hielo. </li><li> La sonda desnaturalizada se añade 5,9 ml de solución de hibridación, que contiene: <ul><li> 3,0 ml de formamida </li><li> 1,5 ml de SSC </li><li> 1,0 ml de H 2 O </li><li> 0,15 ml 50X Denhardt </li><li> 0,10 ml de 20% SDS </li><li> 0,15 ml de esperma de salmón 10mg/ml ADN </li></ul></li><li> La solución de prehibridación se derrama de la botella de rodillo y la solución de hibridación se vierte pulg La botella de rodillos se devuelve a los 42 ° C incubadora y se gira desde hace más de 12 horas. </li><li> La solución de hibridación se derrama de la botella de rodillo. Entonces, la mancha se lavaron 4 veces durante 20 minutos en la botella de rodillos con ~ 150 ml de una solución de lavado que contiene 0,5 X SSC, 0,1% SDS a 42 ° C con rotación. </li><li> Tras el último lavado, la membrana se coloca sobre una toalla de papel hasta que el líquido ha desaparecido visiblemente. La membrana se envuelve en papel de plástico de polivinilo de cloruro y se expone a una pantalla phosphorimager. Nos visualizar y cuantificar la radiactividad con una tormenta de 840 y su software asociado ImageQuant. </li></ol> 4. Los resultados representativos: <img src="/files/ftp_upload/2220/2220fig1.jpg" alt="Figura 1" /> Figura 1. Intermediarios de replicación del plásmido observada en la presencia y ausencia de daño en el ADN inducido por UV. El patrón de migración de PvuII digerido plásmido pBR322 observado por 2D-gel de agarosa-análisis es un diagrama. No repetición de los plásmidos lineales se ejecute como un lineal de 4,4 kb fragmento. Plásmidos replicar la forma en forma de Y las estructuras que migran más lentamente que los fragmentos no replican debido a su mayor tamaño y noforma nlinear. Este patrón de migración en forma de arco que se extiende desde la región lineal hacia el pozo. Después de la irradiación UV, doble-Y o moléculas en forma de X se observan en la región del cono que se desplaza más lentamente que el arco en forma de Y de las moléculas. Un ejemplo del análisis de Southern del gel 2D probaron con marcado con 32P pBR322 se muestra de las células inmediatamente después de la irradiación UV y 15 minutos después de la irradiación UV (reproducido con permiso del editor).

<ol>
	<li>Davis, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Isolation+of+Biochemically+Deficient+Mutants+of+Bacteria+by+Means+of+Penicillin.">The Isolation of Biochemically Deficient Mutants of Bacteria by Means of Penicillin.</a> Proc Natl Acad Sci U S A. 35, 1-10 (1949).</li><li>Courcelle, J., Donaldson, J. R., Chow, K. H., Courcelle, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Damage-Induced+Replication+Fork+Regression+and+Processing+in+Escherichia+coli.">DNA Damage-Induced Replication Fork Regression and Processing in Escherichia coli.</a> Science. 299, 1064-1067 (2003).</li><li>Friedman, K. L., Brewer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+replication+intermediates+by+two-dimensional+agarose+gel+electrophoresis.">Analysis of replication intermediates by two-dimensional agarose gel electrophoresis.</a> Methods Enzymol. 262, 613-627 (1995).</li><li>Spivak, G., Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+damage+and+repair+in+specific+DNA+sequences.">Determination of damage and repair in specific DNA sequences.</a> METHODS: A Companion to Methods in Enzymology. 7, 147-161 (1995).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning - A laboratory manual. , (2001).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvABC+is+required+to+resolve+holliday+junctions+that+accumulate+following+replication+on+damaged+templates+in+Escherichia+coli.">RuvABC is required to resolve holliday junctions that accumulate following replication on damaged templates in Escherichia coli.</a> J Biol Chem. 281, 28811-28821 (2006).</li><li>Donaldson, J. R., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuvAB+and+RecG+Are+Not+Essential+for+the+Recovery+of+DNA+Synthesis+Following+UV-Induced+DNA+Damage+in+Escherichia+coli.">RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli.</a> Genetics. 166, 1631-1640 (2004).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecO+Acts+with+RecF+and+RecR+to+Protect+and+Maintain+Replication+Forks+Blocked+by+UV-induced+DNA+Damage+in+Escherichia+coli.">RecO Acts with RecF and RecR to Protect and Maintain Replication Forks Blocked by UV-induced DNA Damage in Escherichia coli.</a> J Biol Chem. 279, 3492-3496 (2004).</li><li>Belle, J. J., Casey, A., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+DnaB+helicase+leads+to+the+collapse+and+degradation+of+the+replication+fork:+a+comparison+to+UV-induced+arrest.">Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.</a> J Bacteriol. 189, 5452-5462 (2007).</li><li>Chow, K. H., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecBCD+and+RecJ/RecQ+initiate+DNA+degradation+on+distinct+substrates+in+UV-irradiated+Escherichia+coli.">RecBCD and RecJ/RecQ initiate DNA degradation on distinct substrates in UV-irradiated Escherichia coli.</a> Radiat Res. 168, 499-506 (2007).</li><li>Al-Hadid, Q., Ona, K., Courcelle, C. T., Courcelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RecA433+cells+are+defective+in+recF-mediated+processing+of+disrupted+replication+forks+but+retain+recBCD-mediated+functions.">RecA433 cells are defective in recF-mediated processing of disrupted replication forks but retain recBCD-mediated functions.</a> Mutat Res. 645, 19-26 (2008).</li></ol>

Los resultados típicos obtenidos a partir de células de tipo salvaje en presencia y ausencia de daño inducido por UV se muestra en la Figura 1. En la ausencia de daños, aproximadamente el 1% del ADN total de plásmido se puede encontrar en el arco Y cuando las células están creciendo rápidamente en la fase exponencial. Después de la irradiación, un aumento transitorio de las moléculas en forma de Y se observa como las horquillas de replicación bloqueadas se acumulan en los sitios dañados. Los intermediarios ...

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		<table border="1">
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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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<td>An example of the Southern analysis of the 2D gel probed</td>
<td>&nbsp;</td>
<td>GE</td>
<td>G15T8</td>
<td>Yellow Lighting</td>
</tr>
<tr>
<td>15 &mu;watt germicidal lamp</td>
<td>&nbsp;</td>
<td>Sylvania</td>
<td>F20T12/GO</td>
<td>UV Lamp</td>
</tr>
<tr>
<td>Blak-Ray UV Intensity Meter 254nm</td>
<td>&nbsp;</td>
<td>Daigger</td>
<td>EF28195T</td>
<td>UVC photometer</td>
</tr>
<tr>
<td>0.025 &mu;m pore disks</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>VSWP04700</td>
<td>Floating dialysis disks</td>
</tr>
<tr>
<td>PvuII</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>ER0632</td>
<td>Restriction Endonuclease</td>
</tr>
<tr>
<td>Nick-translation kit</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>976776</td>
<td>To make 32P-labeled probe</td>
</tr>
<tr>
<td>Blotting Paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>3030-704</td>
<td>For Southern transfer</td>
</tr>
<tr>
<td>Nylon membrane</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>RPN203S</td>
<td>For Southern transfer</td>
</tr>		</tbody>
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visualization of uv-induced replication intermediates in e. coli using two-dimensional agarose-gel analysis

Replicación inexacta en la presencia de daño en el ADN es el responsable de la mayoría de los reordenamientos celulares y mutagénesis observado en todos los tipos celulares y se cree que estar directamente asociado con el desarrollo del cáncer en los seres humanos. Daños en el ADN, como la inducida por la radiación UV, que limita enormemente la capacidad de replicación de duplicar la plantilla de genómica precisa. Una serie de productos de los genes han sido identificados que son necesarios cuando se encuentra con la replicación de las lesiones del ADN en la plantilla. Sin embargo, un desafío pendiente ha sido la de determinar cómo estas lesiones proceso de las proteínas durante la replicación<em&gt; In vivo</em&gt;. Usando Escherichia coli como un modelo de sistema, se describe un procedimiento en el cual puede ser de dos dimensiones de agarosa-gel de análisis para identificar los intermedios estructurales que surgen en los plásmidos replicar<em&gt; In vivo</em&gt; Después de la radiación UV-inducida por daño en el ADN. Este procedimiento se ha utilizado para demostrar que las horquillas de replicación bloqueadas por el daño inducido por UV someten a una reversión transitoria que se estabiliza por Reca y varios productos de genes asociados con la vía RecF. La técnica demuestra que estos intermediarios de replicación se mantienen hasta el momento que se correlaciona con la eliminación de las lesiones de reparación por escisión de nucleótidos y se reanuda la replicación.

Watch this Scientific Journal Video about Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis at JoVE.com

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Se presenta un procedimiento por el cual puede ser de dos dimensiones de agarosa-gel de análisis para identificar la estructura de los intermediarios de replicación que se producen después de la irradiación UV.

Visualización de intermediarios de replicación inducidos por UV en E. coli Con dos dimensiones-gel de agarosa Análisis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

visualization-uv-induced-replication-intermediates-e-coli-using-two

Research

JoVE Journal

Biology

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

10.6K vistas.  Portland State University. Se presenta un procedimiento por el cual puede ser de dos dimensiones de agarosa-gel de análisis para identificar la estructura de los intermediarios de replicación que se producen después de la irradiación UV.

Video: Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformación de ADN plásmido en E. coli utilizando el método de choque térmico

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Investigación

Biología

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

La evaluación de la motilidad de células madre neurales uso de un gel de agarosa al dispositivo basado en microfluídica

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

La detección de la agregación amiloide de Semi-desnaturalización electroforesis en gel de agarosa-detergente

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Mutagénesis y funcional protocolos de selección de la evolución dirigida de proteínas en E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.
Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.

Visualización de la replicación del ADN en el DT40 modelo de sistema de vertebrados que utilizan la técnica de fibra de ADN

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
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A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Electroforesis en gel de agarosa para la separación de los fragmentos de ADN

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

Fagos mediada por la entrega de apuntado sRNA Construye para derribar la expresión génica en<em&gt; E. coli</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

La producción de norovirus dominios que sobresale en humanos<em&gt; E. coli</em&gt; Para la cristalografía de rayos X

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

La mucina en gel de agarosa de electroforesis: Western Blotting para glicoproteínas de alta peso molecular


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...