This work was supported by the Sogang University Research Grant of 201410036.

1. DNA biotinilazione <ol><li> Biotinilazione di blunt DNA ended utilizzando Terminal transferasi (TdT) Nota: utilizzare T4 DNA (166 kbp), che è un DNA ended smussato. <ol><li> Aggiungere 5 ml di 2,5 mM CoCl 2, 5 ml di 10 × tampone di reazione, 0,5 ml di 10 mM biotina-11-dUTP, 0.5 ml di transferasi terminale (10 unità) e 0,5 ml di T4 DNA (0,5 mcg / ml) alla miscela di reazione. Rendere ad un volume finale di 50 ml aggiungendo 38,5 ml di acqua. </li><li> Incubare la miscela di reazione a 37 ° C per 1 ora. Nota: tempo di reazione totale può produrre DNA a doppia legato, cioè, è possibile che biotins vengono contrassegnati alle due estremità. </li><li> Arrestare la reazione aggiungendo 5 ml di 0,5 M EDTA, pH 8. </li><li> Tenere il tubo a 4 ° C. </li></ol></li><li> Biotinilazione di DNA ended appiccicoso Utilizzando DNA Ligase Nota: utilizzare λ DNA (48,5 kbp), che è un DNA appiccicoso-end. &lt;ol&gt; <li> Aggiungere 1 ml di T4 DNA ligasi, 5 microlitri di 10 × tampone di legatura, 1 ml di 25 ng / ml DNA λ fago, e aggiungere 43 ml di acqua per fare un volume finale di 50 pl. </li><li> Tenere il tubo a 4 ° C. </li></ol></li></ol> 2. funzionalizzato Derivatizzazione Surface Nota:. Per legare una estremità di una molecola di DNA sulla superficie di vetro, un gruppo amminico primario è silanizzato su un vetrino seguita da rivestimento biotina-PEG come mostrato in Figura 1 Il processo PEGylation è importante per l&#39;imaging DNA a singola molecola perché può ridurre significativamente il rumore casuale creato da attaccamento di molecole indesiderate sulla superficie. <ol><li> Piranha Pulizia Nota: Le soluzioni Piranha reagire violentemente con materiali organici, pertanto, deve essere maneggiato con cura, seguendo le linee guida di sicurezza adeguate. <ol><li> Luogo coprioggetto su un politetrafluoroetilene (PTFE) cremagliera, e tenere premuto By PTFE nastro sigillante filo longitudinale, metà sul bordo delle superfici e per metà sul rack. Dopo il confezionamento, lascia un lungo pezzo (~ 5cm) di nastro per la movimentazione della rastrelliera durante la procedura di pulizia. </li><li> Riempire un bicchiere di vetro 1 L con 350 ml di H 2 SO 4 e 150 ml di H 2 O 2 per rendere soluzione piranha in una cappa aspirante. Inserire la griglia in soluzione piranha per 2 ore. </li><li> soluzione Piranha vuoto dal bicchiere, e coprioggetto risciacquo con acqua deionizzata fino a che il pH dell&#39;acqua nel bicchiere raggiunge neutra. Utilizzare strisce di carta pH. </li><li> Sonicare i rack di vetrini in acqua bicchiere contenente, per 30 min. acqua vuota dal bicchiere appena prima che amino silanizzazione. Utilizzare 75 W di potenza sonicazione per pulire e derivatize i substrati di vetro. </li></ol></li><li> Aminosilanization sulla superficie di vetro <ol><li> Aggiungere 2 ml di N - [3- (trimethoxysilyl) propil] etilendiammina e 10 ml di acido acetico glaciale a 200 mldi alcool metilico in un contenitore di polipropilene pulito per la preparazione della soluzione aminosilanization. </li><li> Mettere vetrini puliti nel contenitore in polipropilene. Agitare a 100 rpm e temperatura ambiente per 30 min. </li><li> Sonicare il bicchiere con derivatizzati vetrini per 15 minuti a 75 W. scuoterli a 100 rpm e temperatura ambiente per almeno 30 minuti. </li><li> Svuotare la soluzione dal bicchiere, e risciacquare coprioggetto con attenzione, una volta con alcool metilico, e due volte con alcol etilico. coprioggetto Conservare in alcool etilico e utilizzarli entro due settimane. </li></ol></li><li> PEGylation del vetrino <ol><li> Rendere 10 ml di 0,1 M sodio bicarbonato (NaHCO 3). Filtrare la soluzione con un filtro a siringa da 0,22 micron. </li><li> Sciogliere 2 mg di carbonato di biotina-PEG-succinimidile (biotina-PEG-SC) e 80 mg di PEG-succinimidyl valerato (MPEG-SVG) in 350 ml di NaHCO 3. Utilizzare un tubo di protezione dalla luce. </li><li> Agitare la provetta con forza per 10 sec e centrifugare a 10.000 g per 1 min per rimuovere le bolle. </li><li> Risciacquare un vetrino con acetone, seguito da risciacquo con alcool etilico. Lasciare che il vetrino all&#39;aria asciugare completamente. </li><li> Mettere una goccia (50 ml) di soluzione di PEG sul vetrino pulito. Coprire la goccia delicatamente con un amino silanizzata copertura di slittamento, senza generare eventuali bolle. </li><li> Collocare i vetrini per 3 ore per una notte in un buio, ben livellato camera umida a temperatura ambiente. Utilizzare un titolare di punta della pipetta vuoto come la camera, con acqua sul fondo. Sigillare soluzione residua PEG nel tubo e la tiene a 4 ° C. </li><li> Sciacquare il vetrino PEGylated accuratamente con acqua deionizzata. Conservare in un luogo buio e asciutto fino al momento dell&#39;uso. </li></ol></li></ol> 3. Assemblare una camera di flusso <ol><li> Realizzare un supporto acrilico perforato, come in Figura 2. Assicurarsi che il diametro dell&#39;inserto tubo è 0,762 millimetri e designare un foro è un ingresso e l&#39;altro èuna uscita (figura 2). </li><li> Posizionare nastri biadesivi Nastro adesivo (larghezza 5-6 mm) su un supporto acrilico (Figura 2), allineando perpendicolarmente ad un foro di ingresso e di uscita, senza perturbare fori canale. Utilizzare queste strisce di nastro come pareti multi-canale per rendere camere. Scrub il nastro utilizzando una punta di pipetta. </li><li> Posizionare un vetrino PEGylated sulla parte superiore per fare camere di flusso, con il lato rivolto verso il basso PEGylated. </li><li> Per evitare qualsiasi soluzione fuoriuscita, premere la parte superiore di un coprioggetto sopra la zona in cui vengono inseriti nastri biadesivi. Aggiungere colla quick-dry epossidica per chiudere i bordi della camera in alto e in basso (colore giallo nella figura 2). </li><li> Collegare un breve brano (2,5 cm) di tubo (diametro esterno, OD, 0,042 &quot;) per una siringa a tenuta di gas e sigillare il giunto con colla epossidica. </li><li> Collegare un lungo tubo flessibile (OD: 0.03 &quot;) per il tubo collegato alla siringa e sigillare il giunto con colla epossidica. </li><li> Riempire il tubing collegato alla siringa con acqua deionizzata. Assicurarsi che non vi è alcuna bolla d&#39;aria. </li><li> Inserire il tubo nel foro della camera di flusso sigillato con colla epossidica. </li><li> Mettere una punta gialla (200 microlitri punta), dall&#39;altro foro come un serbatoio. </li></ol> 4. Esempio Caricamento in camera di flusso Nota: neutravidina può essere sostituito con altre proteine ​​avidina come streptavidina. Tutte le reazioni possono essere eseguite a temperatura ambiente, che sono menzionate. Prendere soluzione campione in una punta di colore giallo, e installare la punta con la soluzione sul foro del supporto acrilico (Figura 2b). <ol><li> Impostare la portata della pompa a siringa a 50 ml / min. Carico 20 ml di proteina avidina (25 ug / ml in soluzione T50, Tris 10 mM, NaCl 50 mM, pH 8), e tenerlo per 10 min. </li><li> Carico 20 l di oligonucleotidi biotinilati (100 micron di 1 × TE), e tenerlo per 10 minuti. Se si utilizza transferasi terminale, saltare il caricodi oligonucleotidi. </li><li> Carico 20 ml di soluzione di DNA dal punto 1 nella camera di flusso ad una portata di 10 ml / min, e tenerlo per 30 min. </li><li> Lavare la camera di flusso con 1 × TE, e caricare 40 ml di FP-DBP 10 (~ 80 Nm). </li><li> Osservare DNA sotto un microscopio a fluorescenza con flusso continuo di molecole di colorazione in 1 × TE su una lente obiettivo 60X. Utilizzare 488 nm laser a stato solido per l&#39;eccitazione di FP (eGFP) -DBP. Per la piena tratto di molecole di DNA, si applicano diverse portate a seconda delle lunghezze di DNA utilizzati. Ad esempio, applicare 50 microlitri / min 48,5 kbp di DNA λ, e 100 microlitri / min per 166 kbp di T4 DNA. <ol><li> Per il riciclaggio del titolare acrilico, immergere camere a flusso assemblati in una soluzione detergente per la casa 12. Rimuovere nastri e resina epossidica con una lama di rasoio e strofinare con le mani per prendere completamente fuori. Sbloccare fori con aghi da siringa. Mantenere i titolari in acqua deionizzata fino a nuovo uso. </li></ol></li></ol>

<ol>
	<li>Smith, S. B., Finzi, L., Bustamante, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Mechanical+Measurements+of+the+Elasticity+of+Single+DNA-Molecules+by+Using+Magnetic+Beads.">Direct Mechanical Measurements of the Elasticity of Single DNA-Molecules by Using Magnetic Beads.</a> Science. 258 (5085), 1122-1126 (1992).</li><li>Finkelstein, I. J., Visnapuu, M. -. L., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+imaging+reveals+mechanisms+of+protein+disruption+by+a+DNA+translocase.">Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase.</a> Nature. 468 (7326), 983-987 (2010).</li><li>Doyle, P. S., Ladoux, B., Viovy, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+a+tethered+polymer+in+shear+flow.">Dynamics of a tethered polymer in shear flow.</a> Phys. Rev. Lett. 84 (20), 4769-4772 (2000).</li><li>Perkins, T. T., Quake, S. R., Smith, D. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxation+of+a+Single+DNA+Molecule+Observed+by+Optical+Microscopy.">Relaxation of a Single DNA Molecule Observed by Optical Microscopy.</a> Science. 264 (5160), 822-826 (1994).</li><li>Cai, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+restriction+endonuclease+maps+of+yeast+artificial+chromosomes+created+by+optical+mapping+on+surfaces.">Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.</a> Proc. Natl. Acad. Sci. U.S.A. 92 (11), 5164-5168 (1995).</li><li>Kim, Y., Jo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutravidin+coated+surfaces+for+single+DNA+molecule+analysis.">Neutravidin coated surfaces for single DNA molecule analysis.</a> Chem. Comm. 47 (22), 6248-6250 (2011).</li><li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanochannel+confinement:+DNA+stretch+approaching+full+contour+length.">Nanochannel confinement: DNA stretch approaching full contour length.</a> Lab on a Chip. 11 (10), 1721-1729 (2011).</li><li>Glazer, A. N., Rye, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+dye-DNA+intercalation+complexes+as+reagents+for+high-sensitivity+fluorescence+detection.">Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection.</a> Nature. 359 (6398), 859-861 (1992).</li><li>Graneli, A., Yeykal, C. C., Prasad, T. K., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organized+arrays+of+individual+DNA+molecules+tethered+to+supported+lipid+bilayers.">Organized arrays of individual DNA molecules tethered to supported lipid bilayers.</a> Langmuir. 22 (1), 292-299 (2006).</li><li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+binding+fluorescent+proteins+for+the+direct+visualization+of+large+DNA+molecules.">DNA binding fluorescent proteins for the direct visualization of large DNA molecules.</a> Nucleic Acids Res. , (2015).</li><li>Roy, R., Hohng, S., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+single-molecule+FRET.">A practical guide to single-molecule FRET.</a> Nat Methods. 5 (6), 507-516 (2008).</li><li>Chandradoss, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> J. Vis. Exp. (86), e50549 (2014).</li></ol>

The authors have nothing to disclose.

Qui vi presentiamo una piattaforma per la visualizzazione di molecole di DNA lunghe biotinilati per l&#39;ancoraggio sulle superfici. Abbiamo riportato un approccio per molecole di DNA impastoiati su una superficie rivestita proteina avidina con biotinilato albumina sierica bovina. 6 Nell&#39;approccio in precedenza, abbiamo trovato una questione cruciale del DNA foto-scissione causata da coloranti bis-intercalazione che macchiano molecole di DNA legato alla superficie. Poiché questi fluorofori continuamente...

La visualizzazione di grandi molecole di DNA impastoiati sulle superfici di vetro o perline è stato utilizzato per lo studio delle interazioni DNA-proteina, la dinamica delle proteine ​​su substrato di DNA, 1,2 e fisica dei polimeri. 3,4 Una piattaforma per le grandi molecole di DNA a singolo tethered ha pochi distinta vantaggi rispetto ad altri metodi di immobilizzazione DNA. 5 primo, una grande molecola di DNA legato sulla superficie ha una naturale conformazione random coil senza un flusso di taglio, che è criticamente importante per una proteina di legame al DNA di riconoscere suo sito di legame. In secondo luogo, è molto facile cambiare l&#39;ambiente chimico intorno molecole di DNA per una serie di reazioni enzimatiche in una camera di flusso. In terzo luogo, un flusso di taglio microfluidico induce DNA molecolare estende fino al 100% della lunghezza del profilo completo, che è molto difficile da ottenere con allungamento DNA approcci alternativi come superficie di immobilizzazione 6 e nanochannel confinamento. 7 Un completamente stretched molecola di DNA fornisce anche informazioni di posizione che può essere utile per il controllo dei movimenti enzimatici sulla mappa genomica. Tuttavia, l&#39;approccio tethering DNA ha una lacuna critica che tintura del intercalante come YOYO-1 provoca generalmente molecole di DNA legato ad essere facilmente rotto dalla luce di fluorescenza di eccitazione. Generalmente, grandi molecole di DNA devono essere colorato con un colorante fluorescente per la visualizzazione al microscopio a fluorescenza. A questo scopo, YOYO-1 o altri coloranti della serie TOTO sono utilizzati principalmente perché questi coloranti fluorescenti solo quando intercalare DNA a doppia elica. 8 Tuttavia, è ben noto che bis intercalanti colorante provoca DNA indotta dalla luce foto-scissione, perché della intercalazione di fluorofori. 9 Inoltre, molecole di DNA fluorescente macchiate impastoiati in superficie sono più fragili in quanto i flussi di taglio possono esercitare rottura forze sul liberi di muoversi molecole di DNA. Pertanto, abbiamo sviluppato FP-DBP come un romanzo Ddye proteina NA-colorazione per l&#39;imaging di grandi molecole di DNA impastoiati in superficie. Il vantaggio di utilizzare FP-DBP è che non provoca foto-scissione delle molecole di DNA a cui si lega. 10 Inoltre, FP-DBP non aumenta la lunghezza del profilo del DNA, mentre bis-intercalanti coloranti aumentano la lunghezza del profilo di circa il 33%. Questo metodo di video introduce l&#39;approccio sperimentale per legare grandi molecole di DNA ad una superficie PEG-biotina. La Figura 1 illustra i diversi approcci di legare il DNA con estremità smussata ed estremità appiccicose. Così, questo metodo di colorazione può essere applicato a qualsiasi tipo di molecola di DNA. La Figura 2 illustra una rappresentazione schematica del gruppo camera di flusso che può essere controllata da una pompa a siringa per generare flussi di taglio per allungare molecole di DNA e per caricare chimica ed enzimatica soluzioni. la figura 3 mostra micrografie di molecole di DNA completamente allungato impastoiati sul PEGylatdi superficie ed 11 e colorati con FP-DBP.

<table border="0" cellpadding="0" cellspacing="0" width="925">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">1. DNA Biotinylation</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">1.1) Biotinylation of blunt-end DNA using Terminal Transferase (TdT)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Terminal Transferase</td>
			<td style="width:167px;">New England Biolabs</td>
			<td style="width:133px;">M0315S</td>
			<td>Provided with 10x reaction buffer, 2.5 mM Cobalt chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Biotin-11-dUTP</td>
			<td style="width:167px;">Invitrogen</td>
			<td style="width:133px;">R0081</td>
			<td>Biotin-ddNTP is also available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">T4GT7 Phage DNA</td>
			<td style="width:167px;">Nippon Gene</td>
			<td style="width:133px;">318-03971</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">E6758</td>
			<td>EDTA</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">1.2) Biotinylation of sticky end DNA Using DNA Ligase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">T4 DNA Ligase</td>
			<td style="width:167px;">New England Biolabs</td>
			<td style="width:133px;">M0202S</td>
			<td>Provided with 10x reaction buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">Lambda Phage DNA</td>
			<td style="width:167px;">Bioneer</td>
			<td style="width:133px;">D-2510</td>
			<td>Also available at New England Biolabs</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:925px;">2. Functionalized Surface Derivatization</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">2.1) Piranha Cleaning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Coverslip</td>
			<td style="width:167px;">Marienfeld-Superior</td>
			<td style="width:133px;">0101050</td>
			<td>22x22 mm, No. 1 Thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Teflon rack</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td>Custom Fabrication</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">PTFE Thread Seal Tape</td>
			<td style="width:167px;">Han Yang Chemical Co. Ltd.</td>
			<td style="width:133px;">3032292</td>
			<td>Teflon&#8482; tape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sulferic acid</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">S280823</td>
			<td>H2SO4, 95 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hydrogen peroxide</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">H290423</td>
			<td>H2O2, 35 % in water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Sonicator</td>
			<td style="width:167px;">Daihan Scientific Co. Ltd.</td>
			<td style="width:133px;">WUC-A02H</td>
			<td>Table-top Ultrasonic Cleaner</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">2.2) Aminosilanization on Glass Surface</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">N-[3-(Trimethoxysilyl)propyl] 
			ethylenediamine</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">104884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Glacial Acetic Acid</td>
			<td style="width:167px;">Duksan Chemicals</td>
			<td style="width:133px;">414</td>
			<td>99 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Methyl Alcohol</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">M300318</td>
			<td>99.9 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Polypropylene Container</td>
			<td style="width:167px;">Qorpak</td>
			<td style="width:133px;">PLC-04907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ethyl Alcohol</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">A300202</td>
			<td>99.9 % Purity</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">2.3) PEGylation of the coverslip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sodium Bicarbonate</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syringe Filter</td>
			<td style="width:167px;">Sartorius</td>
			<td style="width:133px;">16534----------K</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Biotin-PEG-SC</td>
			<td style="width:167px;">Laysan Bio</td>
			<td style="width:133px;">Biotin-PEG-SC-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">mPEG-SVG</td>
			<td style="width:167px;">Laysan Bio</td>
			<td style="width:133px;">MPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acetone</td>
			<td style="width:167px;">Jin Chemical Co. Ltd.</td>
			<td style="width:133px;">A300129</td>
			<td>99 % Purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microscope Slides</td>
			<td style="width:167px;">Marienfeld-Superior</td>
			<td style="width:133px;">1000612</td>
			<td>~76x26x1 mm</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">3. Assembling a Flow Chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acrylic Support</td>
			<td style="width:167px;"></td>
			<td style="width:133px;"></td>
			<td>Custom Fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Double-sided Tape</td>
			<td style="width:167px;">3M</td>
			<td style="width:133px;"></td>
			<td>Transparent type</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Quick-dry Epoxy</td>
			<td style="width:167px;">3M</td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Polyethylene Tubing</td>
			<td style="width:167px;">Cole-Parmer</td>
			<td style="width:133px;">06417-11, 06417-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Gas Tight 250 &#181;l Syringe</td>
			<td style="width:167px;">Hamilton</td>
			<td style="width:133px;">81165</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Syringe Pump</td>
			<td style="width:167px;">New Era Pump Systems Inc.</td>
			<td style="width:133px;">NE-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:925px;">4. Sample Loading into Flow Chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Neutravidin</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:133px;">31000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Tris base</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:133px;">T1503-5KG</td>
			<td>Trizma base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:133px;">IX70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">EMCCD Camera</td>
			<td style="width:167px;">Q Imaging</td>
			<td style="width:133px;">Rolera EM-C2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Solid-state Laser (488 nm)</td>
			<td style="width:167px;">Oxxius</td>
			<td style="width:133px;">LBX488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Alconox</td>
			<td style="width:167px;">Alconox Inc.</td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of surface-tethered large dna molecules with a fluorescent protein dna binding peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

La Figura 1 mostra due diversi metodi tethering DNA seconda strutture terminali della molecola di DNA. Figura 1a illustra come i appiccicose molecole di DNA ended sono ibridate con oligonucleotidi biotinilati complementari, che sono immobilizzati sulla superficie PEG avidina-rivestita. Figura 1b mostra l&#39;aggiunta di biotinilato ddNTP o dNTP al gruppo idrossilico 3 &#39;di un DNA ended smussato da transferasi terminale. Abbiamo aggiun...

Watch this Scientific Journal Video about Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide at JoVE.com

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Visualizzazione del Surface-legato Grande DNA molecole con un DNA proteina fluorescente Binding peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

visualization-surface-tethered-large-dna-molecules-with-fluorescent

Research

JoVE Journal

Biology

10.7K Views.  Sogang University. We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows. 

Video: Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizzazione singola molecola di DNA replica con microscopia a fluorescenza

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Ricerca

Biologia

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

Osservazione diretta del DNA Enzimi Replica utilizzando un singolo-molecola di DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA Stabile-isotopica Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

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 ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

Misurazione Traslocazione Peptide in grandi vescicole unilamellari

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Preparazione di DNA-reticolato poliacrilammide idrogel

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

Ridotto gravità ambiente hardware Dimostrazioni di un prototipo miniaturizzato Citofluorimetro e Companion Microfluidic tecnologia di miscelazione

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-base Espressione Proiezione di proteine ​​di membrana in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

Visualizzazione di miniSOG Tagged proteine ​​di riparazione del DNA, in combinazione con Electron spettroscopica Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Visualizzazione del Surface-legato Grande DNA molecole con un DNA proteina fluorescente Binding peptide

Riepilogo

Esplora Altri Video

Capitoli in questo video

Visualizzazione singola molecola di DNA replica con microscopia a fluorescenza

Osservazione diretta del DNA Enzimi Replica utilizzando un singolo-molecola di DNA Stretching Assay

DNA Stabile-isotopica Probing (DNA-SIP)

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

Misurazione Traslocazione Peptide in grandi vescicole unilamellari

Preparazione di DNA-reticolato poliacrilammide idrogel

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

Ridotto gravità ambiente hardware Dimostrazioni di un prototipo miniaturizzato Citofluorimetro e Companion Microfluidic tecnologia di miscelazione

Green Fluorescent Protein-base Espressione Proiezione di proteine di membrana in

Visualizzazione di miniSOG Tagged proteine di riparazione del DNA, in combinazione con Electron spettroscopica Imaging (ESI)

Visualizzazione del Surface-legato Grande DNA molecole con un DNA proteina fluorescente Binding peptide

Riepilogo

Esplora Altri Video

Capitoli in questo video

Visualizzazione singola molecola di DNA replica con microscopia a fluorescenza

Osservazione diretta del DNA Enzimi Replica utilizzando un singolo-molecola di DNA Stretching Assay

DNA Stabile-isotopica Probing (DNA-SIP)

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

Misurazione Traslocazione Peptide in grandi vescicole unilamellari

Preparazione di DNA-reticolato poliacrilammide idrogel

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

Ridotto gravità ambiente hardware Dimostrazioni di un prototipo miniaturizzato Citofluorimetro e Companion Microfluidic tecnologia di miscelazione

Green Fluorescent Protein-base Espressione Proiezione di proteine ​​di membrana in

Visualizzazione di miniSOG Tagged proteine ​​di riparazione del DNA, in combinazione con Electron spettroscopica Imaging (ESI)

Green Fluorescent Protein-base Espressione Proiezione di proteine di membrana in

Visualizzazione di miniSOG Tagged proteine di riparazione del DNA, in combinazione con Electron spettroscopica Imaging (ESI)