Lavoro nel nostro laboratorio è supportato da premio CARRIERA MCB0551798 dalla National Science Foundation e AREA concedere R15GM86839 dal NIGMS-NIH.

1. Crescita e di irradiazione UV. <ol><li> 200μl di una cultura fresco durante la notte contenente il plasmide pBR322 cresciuto in media 1 Davis integrato con 0,4% di glucosio, 0,2% di acidi casamino, e 10 mg / ml timina (media DGCthy) e 100 mg / ml ampicillina è pellet. Il pellet cellulare viene risospeso in medium DGCthy 200μl manca ampicillina e utilizzati per inoculare 20 ml di medium DGCthy. </li><li> Le culture sono cresciute senza selezione ampicillina in una agitazione incubatore a 37 ° C ad un diametro esterno 600 di 0,5 (~ 5 x 10 8 cellule / ml). Crescita senza ampicillina evita selezione contro intermedi replica anormale o improduttivi che possono sorgere in alcuni mutanti. Inoltre, se si utilizza la luce UV per indurre danni, la rimozione del ampicillina da parte dei media è necessario perché assorbe fortemente a queste lunghezze d&#39;onda e scudi le cellule, riducendo la dose efficace di UV. </li><li> Lavorare sotto le luci gialle, la cultura è posto in un piatto di 15 centimetri di diametro Petri su una piattaforma rotante per l&#39;agitazione. Le nostre culture sono posti ad una distanza da un 15-watt lampada germicida che produce un tasso di esposizione di circa 1 J/m2/sec, che viene misurata con un fotometro UVC. Le culture sono irradiati con 50 J/m2 e poi messo immediatamente in agitazione i 37 ° C incubatore per tutta la durata dell&#39;esperimento. Questa dose produce, in media, 1 ciclobutano dimero pirimidina ogni 4,5 kb di ssDNA. La luce gialla impedisce fotoriattivazione-inversione di dimeri pirimidinici ciclobutano da fotoliasi. </li></ol> 2. Isolamento del DNA. <ol><li> A volte, quando intermedi replica sono da esaminare, una aliquota 0,75 ml di cultura viene inserito in 0,75 ml di ghiaccio freddo NET30 Buffer (100 mM NaCl, 10 mM Tris, pH 8.0, 30 mM EDTA) e messo in ghiaccio fino alla fine del la durata del corso. Noi di solito un decorso di tempo 90 minuti, con i campioni esaminati a 0, 15, 30, 45, 60 e 90 minuti. L&#39;EDTA e la temperatura fredda servono a bloccare efficacemente la replicazione e riparazione per escissione dei nucleotidi. </li><li> Ogni campione è poi pellet, risospeso in 150 ml di 1,5 mg / ml di lisozima e 0,2 mg / ml RNaseA a TE (10 mM Tris, pH 8.0, 1 mM EDTA), e lisato a 37 ° C per 20 min. In questo momento, 10μl di proteinasi K (10 mg) e 10μl del 20% sarkosyl sono aggiunti e l&#39;incubazione è permesso di proseguire per 1 ora. Proteinasi K e sarkosyl aiutare a liberare frammenti di DNA che possono essere associati con la membrana o proteine ​​di prima estrazione fenolo si verifica. Dal DNA replicazione attiva è spesso legato a complessi proteici o membrana, questo aiuta ad aumentare la resa di frammenti di replica che sono recuperati. </li><li> I campioni vengono poi estratti con l&#39;aggiunta di 2 volumi di fenolo per ogni campione e i tubi sono capovolta con cautela per 5 minuti. Quindi 2 volumi di cloroformio / alcool isoamilico (24 / 1) sono aggiunte ei tubi sono capovolta con cautela per 5 minuti. </li><li> I campioni vengono centrifugati a 14.000 rpm in una microcentrifuga per 5 minuti e la fase acquosa superiore di ogni campione è stato rimosso, e collocato in una nuova provetta. Poi 4 volumi di cloroformio / alcool isoamilico (24 / 1) sono aggiunti, i tubi sono capovolta con cautela per 5 minuti, e centrifugato di nuovo a 14.000 rpm per 5 minuti. </li><li> Il piano, in fase acquosa ogni campione viene poi dializzati per 1 ora di spotting 100μl di ciascun campione su un disco Whatman 47 millimetri 0,025 micron pori che galleggia su 250 ml di 0,1 X. TE in un bicchiere. Perché le strutture replicare con le regioni singolo filamento o punti di ramificazione sono più sensibili al taglio, di solito tagliare le nostre puntali con una lama di rasoio per rendere la bocca più ampia e minimizzare forze di taglio. In generale, pipettaggio dovrebbero essere ridotte al minimo fino a dopo il DNA è stato digerito con enzimi di restrizione. </li><li> Ogni campione viene digerito con PvuII (New England Biolabs) che linearizza il plasmide appena a valle l&#39;origine di replicazione. </li><li> Prima di caricare nel gel di agarosio, 100μl cloroformio e 20μl di colorante di caricamento 6X che contiene bromofenolo Cyanol Blu e xilene sono aggiunti ad ogni campione e misti. Poi, 40μl della fase acquosa contenente il colorante di caricamento viene caricata nel gel. Per ridurre al minimo i pregiudizi strutturali, manufatti strutturali, e di taglio del DNA, questa procedura di isolamento non prevede alcuna misura per arricchire per le regioni singolo filamento, precipitare, o concentrato i campioni di DNA segue la lisi cellulare. </li></ol> 3. Gel 2D e analisi del sud. <ol><li> Il 2-dimensionale agarosio-gel analisi viene modificato da 3. Per la dimensione 1 °, i campioni di DNA ristretto vengono eseguiti attraverso un gel di agarosio 0,4% in TBE 1X a 1V/cm. Un litro di soluzione di TBE 10X magazzino contiene: <ul><li> 108 g Tris Base </li><li> 55 g di acido borico </li><li> 40 ml di EDTA 0,5 M (pH 8,0) </li></ul></li><li> Un Hind lambda marcatore dimensioni III è caricato nel vicolo, poi i campioni sono stati caricati in ogni altra corsia. Noi di solito eseguire il primo dimension ~ 12-15 ore. Salto di corsia rende più semplice per tagliare fuori le corsie di lanciare nella seconda dimensione. La bassa tensione e bassa percentuale gel serve per separare i frammenti di DNA in primo luogo sulla base delle dimensioni. </li><li> Per la seconda dimensione, corsie gel sono fette fuori del gel prima dimensione usando un coltello da macellaio di grande. Prima corsia contenente il marcatore lambda Hind III possono essere colorati con bromuro di etidio. Utilizzando il lambda marcatore Hind III come coltura guida, e scartare la regione del gel che è al di sotto dove il plasmide linearizzato è previsto per l&#39;esecuzione. In queste condizioni, troviamo che pBR322 linearizzato corre leggermente al di sopra del cyanol xilene macchia. </li><li> Per lanciare la seconda dimensione, ogni corsia è posto orizzontalmente nella parte superiore di un gel caster vuoto. Una soluzione di 1,0% di agarosio in TBE 1X è preparato e raffreddato a 55 ° C. A questo punto, la soluzione gel viene versato per lanciare la seconda dimensione, avendo cura di coprire completamente le fette di gel. Una volta che il gel ha creato, è gestito in 6.5V/cm in una unità di elettroforesi che permette il buffer di ricircolo. Noi di solito eseguire la seconda dimensione ~ 5,5-7 ore. L&#39;alta tensione e alta cento gel separa in modo efficace i frammenti di DNA in base alla loro forma, così come le dimensioni. Forme non lineari eseguire più lentamente attraverso la seconda dimensione. </li><li> A seguito di elettroforesi, il gel viene poi sciacquato in H 2 O, lavate due volte in 400 ml di acido cloridrico 0.25M per 15 minuti, sciacquati con H 2 O, e quindi lavato due volte in 400 ml di NaOH 0.4M per 30 minuti. L&#39;acido lava servono a parzialmente nick le molecole di DNA in frammenti più piccoli che il trasferimento in modo più efficiente. Questo passaggio è necessario a causa delle grandi dimensioni e forme insolite di intermedi replicazione del DNA. </li><li> Il DNA nel gel viene poi trasferito in un Hybond N + membrana di nylon 4. Usiamo un sistema di trasferimento verso il basso alcali con NaOH 0.4M. In breve, due fogli di carta assorbente imbevuta di 0.4M NaOH sono disposti su un grande pila di tovaglioli di carta. La membrana di nylon è anche intriso di NaOH 0.4M e posto sulla parte superiore della carta assorbente. Il gel viene poi accuratamente a strati in cima a questa, seguita da un altro pezzo di carta assorbente, bagnato nella soluzione di NaOH. Infine, un lungo pezzo di carta assorbente bagnata è stratificato nella parte superiore e le sue due estremità sono posti in una specialità contenente 1 L di soluzione 0.4M di NaOH a servire come uno stoppino. Noi di solito lasciare che il trasferimento del DNA per ore 6-12. </li><li> La membrana di nylon è stato rimosso, lavato 20-30 secondi a 5X Buffer SSC, e messo in una bottiglia rullo ibridazione contenente soluzione 10.5ml Prehybridization. Poi, la membrana viene incubata a 42 ° C con rotazione per più di 6 ore. Prehybridization soluzione: <ul><li> 5,0 ml formamide </li><li> 0,5 ml 20% SDS </li><li> 2,5 ml 20X SSC * </li><li> * 2,0 ml di 50X Denhardt </li><li> 0,5 ml di sperma DNA Salmon (10mg/ml) * Ricette per SSC e Denhardt si possono trovare in 5 </li></ul></li><li> Durante il periodo prehybridization, 1 mg di plasmide pBR322 è marcato con 32P dalla traduzione nick secondo il protocollo fornito da alfa Roche utilizzando l&#39;etichetta [32 P]-dCTP. La sonda radiomarcata (100μl) è denaturato incubando a 98 ° C per 10 minuti in una provetta tappo a vite, e poi messo immediatamente in ghiaccio. </li><li> La sonda denaturato viene aggiunta a 5,9 ml di soluzione di ibridazione, che contiene: <ul><li> 3,0 ml formamide </li><li> 1,5 ml SSC </li><li> 1,0 ml H 2 O </li><li> 0,15 ml di 50X Denhardt </li><li> 0,10 ml 20% SDS </li><li> 0,15 ml 10mg/ml Salmone DNA dello sperma </li></ul></li><li> La soluzione prehybridization viene versata dalla bottiglia rullo e la soluzione di ibridazione viene versata in bottiglia Il rullo viene quindi restituito ai 42 ° C incubatore e ruotato per più di 12h. </li><li> La soluzione di ibridazione viene versata dalla bottiglia rullo. Poi, la macchia è lavata 4 volte per 20 minuti in bottiglia rullo con circa 150 ml di una soluzione di lavaggio contenente 0.5X SSC, 0,1% SDS a 42 ° C con rotazione. </li><li> Dopo l&#39;ultimo lavaggio, la membrana è posta su un tovagliolo di carta fino a quando il liquido è visibilmente scomparso. La membrana viene poi avvolto in polivinile cloruro di pellicola trasparente e esposti a uno schermo phosphorimager. Noi visualizzare e quantificare la radioattività utilizzando una tempesta 840 e il suo software associati ImageQuant. </li></ol> 4. Rappresentante dei risultati: <img src="/files/ftp_upload/2220/2220fig1.jpg" alt="Figura 1" /> Figura 1. Intermedi di replicazione plasmide osservato in presenza e in assenza di raggi UV-indotto danni al DNA. Il modello migratorio di PvuII digerito pBR322 plasmide osservato da 2D-gel di agarosio-analisi è diagramma. Plasmidi lineari non replicare eseguito come lineare da 4,4 kb frammento. Plasmidi replicare forma ad Y strutture che migrano più lentamente i frammenti a causa della loro maggiore dimensione non replicare e nonforma nlinear. Questo modello migratorio forma un arco che si estende fuori dalla regione lineare verso il pozzo. In seguito a irradiazione UV, doppia-Y o X a forma di molecole sono osservate nella regione del cono che migra più lentamente l&#39;arco a forma di Y molecole. Un esempio di analisi del sud del gel 2D sondato con marcato con 32P pBR322 è indicato per le cellule immediatamente dopo l&#39;irradiazione UV e 15 minuti dopo l&#39;irradiazione UV (riprodotto con il permesso dell&#39;editore).

<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>

Nessun conflitto di interessi dichiarati.

I risultati tipici ottenuti da cellule di tipo selvatico in presenza e in assenza di danno indotto dai raggi UV sono mostrati in figura 1. In assenza di danni, ~ 1% del DNA totale plasmide si possono trovare nell&#39;arco Y quando le cellule sono in rapida crescita in fase esponenziale. A seguito di irradiazione, un aumento transitorio delle molecole a forma di Y si osserva come le forcelle replica bloccato accumulano nei siti danneggiati. La forma di X intermedi replica anche transitoriamente si accumulano e persistono...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<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>
		</table>
		</div>

visualization of uv-induced replication intermediates in e. coli using two-dimensional agarose-gel analysis

Replica inesatte in presenza di danno al DNA è responsabile della maggior parte dei riarrangiamenti cellulare e mutagenesi osservato in tutti i tipi di cellule ed è ampiamente creduto di essere direttamente associati con lo sviluppo del cancro negli esseri umani. Danno al DNA, come quella indotta da radiazioni UV, limita fortemente la capacità di replicazione di duplicare il modello di genomica con precisione. Un certo numero di prodotti genici sono stati identificati, che sono necessari quando la replicazione incontra le lesioni del DNA nel modello. Tuttavia, una sfida che rimane è stato quello di determinare come queste lesioni processo di proteine ​​durante la replica<em&gt; In vivo</em&gt;. Utilizzando Escherichia coli come sistema modello, si descrive una procedura in cui bidimensionale agarosio-gel analisi può essere utilizzato per identificare gli intermedi strutturali che sorgono su plasmidi replicare<em&gt; In vivo</em&gt; Segue indotte dai raggi UV danno al DNA. Questa procedura è stata utilizzata per dimostrare che le forcelle replica bloccato dal danno indotto dai raggi UV subiscono un&#39;inversione transitoria che è stabilizzata da Reca e dei prodotti genici diversi relativi alla via RECF. La tecnica dimostra che questi intermedi replica vengono mantenute fino a un tempo che si correla con la rimozione delle lesioni da riparazione per escissione dei nucleotidi e riprende la replica.

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

Vi presentiamo una procedura che bidimensionale agarosio-gel analisi può essere utilizzato per identificare la struttura degli intermedi di replica che si verificano dopo irradiazione UV.

Visualizzazione dei raggi UV-indotta Intermedi replica in E. coli Utilizzando bidimensionale agarosio-gel Analisi

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 Views.  Portland State University. Vi presentiamo una procedura che bidimensionale agarosio-gel analisi può essere utilizzato per identificare la struttura degli intermedi di replica che si verificano dopo irradiazione 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.

Trasformazione del DNA plasmidi in E. coli con il metodo Heat Shock

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 ...

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Biologia

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.

Valutare motilità delle cellule staminali neurali L&#39;utilizzo di un gel a base di Agarose dispositivo a microfluidi

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.

Lo screening per aggregazione amiloide di semi-denaturazione detergente-Agarosio elettroforesi su gel

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.

Protocolli di selezione mutagenesi e funzionale per l&#39;evoluzione Regia di proteine ​​in 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.

Visualizzazione della replicazione del DNA nei vertebrati DT40 sistema modello utilizzando la tecnica del DNA Fiber

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.

Elettroforesi su gel agarosio per la separazione di frammenti di DNA

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.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<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.

Fago-mediata consegna di mirata sRNA Costruisce per abbattere l&#39;espressione genica in<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).

Produzione di Norovirus umani sporgenti Domini in<em&gt; E. coli</em&gt; Per la cristallografia a raggi 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.

Mucina Agarosio elettroforesi su gel: Western Blotting per glicoproteine ​​ad alto peso molecolare


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