Name: dna staining method based on formazan precipitation induced by blue light exposure
Uploaded: 2018-01-28T14:00:00.000+00:00
Duration: 8 min 11 s
Description: Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

Questo lavoro è stato supportato da Fondo Nacional de Desarrollo y Científico Tecnológico (Fondecyt), Cile, progetto 11130263 (in senso orario), progetto CONICYT + NERC + Programa de Colaboración Internacional PCI-PII20150073 (in senso orario) e U-inicia dal de Vicerrectoría Investigación Universidad de Chile (in senso orario).
Grazie a Tatiana Naranjo-Palma per l'aiuto nella fase iniziale di questo progetto, Dr. Jorge Babul e Diego Quiroga-Roger per utile discussione, Robert Lesch per rivedere il manoscritto e la Dirección de extensión de la Facultad de Ciencias Químicas y Farmacéuticas da Universidad de Chile. Grazie anche alla Marcelo Pérez per il video editing e Neil Stevens per la correzione del testo ed Errol Watson per fornire la voce fuori campo.

1. preparazione ed esecuzione del Gel
<ol><li>Preparare il non-denaturante 12% gel di poliacrilammide gel utilizzando la ricetta di elettroforesi del Gel di sodio dodecil solfato-poliacrilammide (SDS-PAGE) Trovati a Sambrook e Russell14 ma utilizzando acqua invece di SDS.<ol>
<li>Per preparare 5 mL di gel di risoluzione, utilizzare 1,7 mL di acqua distillata, 2 mL di acrilammide/bis acrilammide (30/0,8% w/v), 1,3 mL di 1,5 M Tris-HCl pH 8,8, 0,05 mL di 10% ammonio persolfato e 0,002 mL di TEMED.</li>
		<li>Per preparare 2 mL di gel d'impilamento utilizzare 1,5 mL di acqua, 0,33 mL di acrilammide/bis acrilammide (30/0,8% w/v), 0,25 mL 1 M Tris pH 6.8, 0,02 mL 10% di persolfato di ammonio e 0,002 mL di TEMED. Le dimensioni del gel sono cm 7,3 x 8,2 x 0,075 cm (l x w x d). Nota: Utilizzare un pettine di ben 15 di 0,75 mm di spessore per la migliore sensibilità.</li>
	</ol>
</li>
	<li>Aggiungere la quantità appropriata di 6x loading buffer e campione di DNA in una provetta da microcentrifuga da 1,5 mL (ad es. uso 1 µ l di tampone di caricamento x 6 a 5 µ l di campione di DNA).</li>
	<li>Posto il gel nella camera di elettroforesi verticale.<ol>
<li>Riempire la camera con 750 mL di 1 x TAE buffer per eseguire 2 gel. Per preparare 1 L di 50 x TAE, utilizzare 442 g di Tris base, 57,1 mL di acido acetico glaciale e 100 mL di 0,5 M EDTA, pH 8. Aggiungere acqua distillata fino ad un volume finale di 1 L.</li>
		<li>Caricare i campioni nel gel. Esegua il gel a 100 V per 2 h.</li>
	</ol>
</li>
	<li>In alternativa, preparare un gel di agarosio x TAE 1 utilizzando la ricetta che trovata Sambrook14.<ol>
<li>Aggiungere 300 mL di 1 x TAE alla camera di elettroforesi orizzontale. Esegua il gel per 45 min a 100 V.</li>
	</ol>
</li>
</ol>2. NBT-SG ho colorazione (Figura 1).
<ol><li>
Gel di acrilamide che macchia.
	<ol>
<li>Preparare le soluzioni: 0,1% p/v NBT e 1.2 x SG mi. Nota: È possibile utilizzare il bromuro di etidio, GelRed e oro SYBR anziché SG ho, ma ho avuto la migliore performance di SG.</li>
		<li>Versare 16,75 mL di 1.2 x SYBR ho verde soluzione in un recipiente di dimensioni adeguate. Nota: Abbiamo usato il coperchio di una scatola di punta della pipetta come un contenitore, e questi volumi sono calcolati per coprire il gel utilizzando questo contenitore; le sue dimensioni sono cm 11,6 x 7,6 x 2,6 cm (l x w x d).</li>
		<li>Posto il gel nella soluzione. Aggiungere 3,25 mL di soluzione NBT 0,1% p/v di dare 0,2 mM NBT e 1x (equivalente a 2,0 µM) SG ho concentrazioni finali.</li>
		<li>Sigillare il contenitore con pellicola di plastica e avvolgere completamente il contenitore con carta stagnola per bloccare qualsiasi luce ambiente. Nota: La pellicola di plastica è utilizzata per evitare schizzi e per evitare la reazione dell'estruso in alluminio con la soluzione colorante.</li>
		<li>Agitare per 25 minuti su agitatore orbitale a 60 giri/min.</li>
		<li>Rimuovere il coperchio di plastica e alluminio. Esporre alla luce solare per 90 min massimo. Controllare ogni 5 minuti finché non viene visualizzata la banda di interesse.</li>
		<li>Per utilizzare la luce blu, preparare un protoboard con 3 diodi emettitori di luce (LED) e posizionare i LED circa 5 mm dalla superficie del gel (controllare la macchiatura continuamente, come il tempo necessario dipenderà l'intensità della sorgente luminosa LED).</li>
	</ol>
</li>
	<li>
Macchiatura del Gel dell'agarosi.
	<ol>
<li>Dopo preparazione e l'esecuzione di un 1% di agarosio gel come descritto nelle sezioni 1.4 (le dimensioni sono 1 x 6,2 cm x 7 cm; l x l x p), seguire la stessa procedura come in 2.1 con queste modifiche:</li>
		<li>Versare 41,9 mL di 1.2 x SG ho soluzione nel coperchio di una casella di punta della pipetta; le sue dimensioni sono cm 11,6 x 7,6 x 2,6 cm (l x w x d). Aggiungere 8,1 mL di soluzione 0,1% NBT.</li>
		<li>Sigillare il contenitore con pellicola di plastica e poi avvolgere completamente il contenitore con carta stagnola per bloccare qualsiasi luce ambiente.</li>
		<li>Agitare per 1 h su agitatore orbitale a 60 giri/min.</li>
	</ol>
</li>
</ol>3. analisi dei dati.
<ol><li>
NBT-SG Ho curve di calibrazione.
	<ol>
<li>Ottenere immagini con gel di scansione a 1200 dpi.</li>
		<li>Aprire il software per analisi di immagine. Raddrizzare le immagini utilizzando un editor di immagini. Eseguire una densitometria di immagine.</li>
		<li>Calcolare l'area sotto la curva. Preparare un grafico del segnale/segnaleMax contro concentrazione di DNA.</li>
	</ol>
</li>
</ol>4. DNA recupero.
<ol><li>Preparare un gel di agarosio 1% in 1 x TAE usando la ricetta trovato in Sambrook14.</li>
	<li>Aggiungere 300 mL di 1 x TAE alla camera di elettroforesi orizzontale. Caricare 4 µ l di 100 ng / µ l pDJ10015 plasmide e DNA ladder seguendo lo schema dato in Figura 2. Esegua il gel per 1 h a 100 V.</li>
	<li>Contrassegnare e pesano due provette microcentrifuga di 2 mL.</li>
	<li>Tagliare il gel nel mezzo al fine di avere due metà identiche. Ognuno dovrebbe contenere la scala e il plasmide di esempio per confrontare le diverse tecniche di colorazione. Una rappresentazione schematica è nella Figura 2.</li>
	<li>Macchia una metà con NBT-SG ho come al punto 2.<ol>
<li>Tagliare la banda di plasmide usando un bisturi pulito. Salvare il pezzo di gel in una provetta e pesare.</li>
	</ol>
</li>
	<li>Macchia l'altra parte del gel con SG I con 50 mL di 1 x SG ho soluzione e agitare lentamente (60 giri) il gel per 1 h nel buio.<ol>
<li>Posizionare il gel su un transilluminatore ed esporre per 2 min taglio fuori della band di plasmide usando un bisturi pulito.</li>
		<li>Salvare il pezzo di gel nell'altro tubo e pesare. Nota: Il protocollo può essere messo in pausa qui mantenendo le provette a-20 ° C.</li>
	</ol>
</li>
	<li>Effettuare il plasmide estrazione utilizzando un kit di estrazione del gel commerciale.</li>
</ol>5. plasmide normalizzazione.
<ol><li>In una provetta da microcentrifuga da 1,5 mL, aggiungere 10 µ l di Estratto del plasmide e 60 µ l di tipo I acqua. Mix e trasferire a una cuvette di quarzo 60 µ l o utilizzare 2 µ l direttamente in un Nanodrop.</li>
	<li>Misurare gli spettri di assorbimento tra 230 a 320 nm. Calcolare la concentrazione di plasmide del campione utilizzando la seguente formula: <img alt="Equation 1" src="/files/ftp_upload/56528/56528eq1v3.jpg"> dove D è il fattore di diluizione applicato (in questo caso è 70/10), Ax è assorbimento a x nm e 50 è il fattore di conversione a µ g/mL per dsDNA.</li>
	<li>Diluire i campioni a 9.1 ng / µ l.</li>
	<li>Trasformare cellule competenti di Escherichia coli (Escherichia coli) (per esempio oro XL10) seguendo la procedura trasformazione di Inoue16 utilizzando 5 µ l di campione diluito.</li>
	<li>Contare il numero di colonie sui piatti Petri e notare la diluizione. Calcolare l'unità di formatura Colonia (CFU) con la seguente formula17: <img alt="Equation 2" src="/files/ftp_upload/56528/56528eq2v2.jpg"> dove D è il fattore di diluizione applicato dalla coltura iniziale.</li>
	<li>Eseguire un test t spaiato.</li>
</ol>

<ol>
	<li>LePecq, J. B., Paoletti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescent+complex+between+ethidium+bromide+and+nucleic+acids:+physical-chemical+characterization.">A fluorescent complex between ethidium bromide and nucleic acids: physical-chemical characterization.</a> J. Mol. Biol. 27 (1), 87-106 (1967).</li><li>Singer, V. L., Lawlor, T. E., Yue, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+SYBR+Green+I+nucleic+acid+gel+stain+mutagenicity+and+ethidium+bromide+mutagenicity+in+the+Salmonella/mammalian+microsome+reverse+mutation+assay+(Ames+test).">Comparison of SYBR Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test).</a> Mutat Res. 439 (1), 37-47 (1999).</li><li>Xin, X., Yue, S., Wells, K. S., Singer, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SYBR+Green-I:+a+new+fluorescent+dye+optimized+for+detection+picogram+amounts+of+DNA+in+gels.">SYBR Green-I: a new fluorescent dye optimized for detection picogram amounts of DNA in gels.</a> Biophys. J. 66 (A150), (1994).</li><li>Flores, N., Valle, F., Bolivar, F., Merino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+DNA+from+agarose+gels+stained+with+methylene+blue.">Recovery of DNA from agarose gels stained with methylene blue.</a> Biotechniques. 13 (2), 203-205 (1992).</li><li>Yang, Y. I., Jung, D. W., Bai, D. G., Yoo, G. S., Choi, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterion-dye+staining+method+for+DNA+in+agarose+gels+using+crystal+violet+and+methyl+orange.">Counterion-dye staining method for DNA in agarose gels using crystal violet and methyl orange.</a> Electrophoresis. 22 (5), 855-859 (2001).</li><li>Yang, Y. I., 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=Detection+of+DNA+using+a+visible+dye,+Nile+blue,+in+electrophoresed+gels.">Detection of DNA using a visible dye, Nile blue, in electrophoresed gels.</a> Anal. Biochem. 280 (2), 322-324 (2000).</li><li>Santill&#225;n-Torres, 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=A+novel+stain+for+DNA+in+agarose+gels.">A novel stain for DNA in agarose gels.</a> Trends Genet. 9 (2), 40 (1993).</li><li>Adkins, S., Burmeister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+DNA+in+agarose+gels+as+migrating+colored+bands:+applications+for+preparative+gels+and+educational+demonstrations.">Visualization of DNA in agarose gels as migrating colored bands: applications for preparative gels and educational demonstrations.</a> Anal. Biochem. 240 (1), 17-23 (1996).</li><li>Cong, W. T., 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=A+visible+dye-based+staining+method+for+DNA+in+polyacrylamide+gels+by+ethyl+violet.">A visible dye-based staining method for DNA in polyacrylamide gels by ethyl violet.</a> Anal. Biochem. 402 (1), 99-101 (2010).</li><li>Altman, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrazolium+salts+and+formazans.">Tetrazolium salts and formazans.</a> Prog. Histochem. Cytochem. 9 (3), 1-56 (1976).</li><li>Nineham, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+of+formazans+and+tetrazolium+salts.">The chemistry of formazans and tetrazolium salts.</a> Chem. Rev. 55 (2), 355-483 (1955).</li><li>Crandall, I. E., Zhao, B., Vlahakis, J. Z., Szarek, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+imidazole-,+imidazolium,+and+tetrazolium-containing+compounds+with+DNA.">The interaction of imidazole-, imidazolium, and tetrazolium-containing compounds with DNA.</a> Bioorg. Med. Chem. Lett. 23 (5), 1522-1528 (2013).</li><li>Paredes, A. J., 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=New+visible+and+selective+DNA+staining+method+in+gels+with+tetrazolium+salts.">New visible and selective DNA staining method in gels with tetrazolium salts.</a> Anal. Biochem. 517, 31-35 (2017).</li><li>Green, M. R., Sambrook, J. <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. , (2012).</li><li>Hansen, W., Garcia, P. 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Un sensibile e romanzo DNA che macchia il metodo basato su riduzione di NBT e l'uso di SG mi è stato presentato in questo articolo. Il passaggio fondamentale in questo protocollo è il tempo di incubazione del gel in NBT-SG ho soluzione e la concentrazione di NBT. Il tempo di colorazione per i gel dell'agarosi è più per i gel di acrilammide a causa del maggiore spessore del gel dell'agarosi. Questo metodo non funziona bene in presenza di SDS come NBT precipita a contatto con esso. Se il gel ottiene uno sfondo viola, s...

Con gli avanzamenti nella biochimica e biologia molecolare, gli studi che coinvolgono il DNA hanno richiesto le tecniche migliori per analizzare il DNA. Il primo metodo per la colorazione del DNA era macchiatura d'argento, che è molto sensibile ma manca selettività e non consente il recupero del campione. In seguito, lo sviluppo di macchiatura del DNA fluorescente permesso la quantificazione selettiva del DNA con la possibilità di recupero del campione. Uno dei primi coloranti fluorescenti utilizzati per la quantificazione del DNA era etidio bromuro1, che è mutageno2. Tuttavia, ora ci sono migliorate le tinture fluorescenti che sono più sicuri e più sensibili, come GelRed e SYBR Green ho (SG ho)3; ma tutti questi coloranti fluorescenti richiedono l'uso di un transilluminatore (UV) ultravioletto o fluorimetro.
Ci sono altre tecniche per visibilmente la colorazione del DNA, come il blu di metilene4 e viola di cristallo5,6,7,8,9, ma tutti questi soffre di ridotta sensibilità e selettività. I sali di tetrazolio sono composti organici suscettibile di riduzione e quando questo accade formano un insolubile e colorata formazan precipitare10,11. Recentemente, alcuni sali di tetrazolio bivalente hanno dimostrati di legarsi al DNA a causa della loro positiva tetrazolium anelli12.
In una recente pubblicazione13, è stata proposta una nuova tecnica visibile di macchia e quantificare il DNA in gel di poliacrilammide, mediante la riduzione di un sale di tetrazolio bivalente chiamato nitro blu di tetrazolio (NBT) e tinture fluorescenti come il bromuro di etidio, GelRed, SYBR Green io e SYBR Gold. Questa reazione ha lavorato in presenza di luce blu o luce del sole e ha permesso il recupero di campione con una migliore qualità rispetto all'uso di SG I con un transilluminatore UV. L'obiettivo di questa carta è di fornire un protocollo dettagliato della tecnica colorazione utilizzando tetrazolium sali.

<table><tbody><tr><td>Nitro blue tetrazolium</td><td>Gold Biotechnology</td><td>298-83-9</td><td>reagent used in the staining</td></tr><tr><td>SYBR Green I 10000X</td><td>Thermo-Fisher</td><td>S7563</td><td>reagent used in the staining</td></tr><tr><td>Gel extraction kit</td><td>QIAGEN</td><td>28704</td><td>used to extract plasmid from the gel in integrity assay</td></tr><tr><td>DNA ladder</td><td>Thermo-Fisher</td><td>SM 1331</td><td>used to make calibration curves and as reference to cut band in integrity assay</td></tr><tr><td>Agar Agar</td><td>Merck</td><td></td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>sodium chloride</td><td>Merck</td><td>1064045000</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>yeast extract</td><td>Becton, Dickinson and Company</td><td>212750</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>peptone</td><td>Merck</td><td>72161000</td><td>used to make LB-ampicillin culture plates&amp;nbsp;</td></tr><tr><td>TEMED</td><td>AMRESCO</td><td>761</td><td>used to make polyacrylamide gels</td></tr><tr><td>ammonium persulfate</td><td>Calbiochem</td><td>2310</td><td>used to make polyacrylamide gels</td></tr><tr><td>acrylamide</td><td>invitrogen</td><td>15512-023</td><td>used to make polyacrylamide gels</td></tr><tr><td>bisacrylamide</td><td>SIGMA</td><td>146072</td><td>used to make polyacrylamide gels</td></tr><tr><td>Tris-base</td><td>Merck</td><td>1083821000</td><td>used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer</td></tr><tr><td>EDTA</td><td>J.T. Baker</td><td>8993-01</td><td>used to make TAE buffer</td></tr><tr><td>Acetic acid</td><td>Merck</td><td>1,000,632,500</td><td>used to make TAE buffer</td></tr><tr><td>agarose</td><td>Merck</td><td>16802</td><td>used to make TAE buffer</td></tr><tr><td>spectrophotometer</td><td>Tecan</td><td>infinite 200 pro</td><td>used to quantify DNA plasmid</td></tr><tr><td>water purificatiom unit</td><td>Merck</td><td>Elix 100</td><td>use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions\</td></tr><tr><td>distilled water</td><td></td><td>-</td><td>used in gels, culture medium, stainings and general solutions</td></tr><tr><td>HCl</td><td>J.T. Baker</td><td>9535-03</td><td>used to make non-denaturing polyacrylamide gel tris-HCl buffer</td></tr><tr><td>6X DNA loading dye</td><td>Thermo-Fisher</td><td>R0610</td><td></td></tr><tr><td>5 mm Blue LED</td><td>Jinyuan electronics</td><td>SP-021</td><td>light source used in integrity assay</td></tr><tr><td>protoboard</td><td>KANDH</td><td>KH102</td><td>light source support and circuit&amp;nbsp;</td></tr><tr><td>9 V battery</td><td>Duracell</td><td>-</td><td>used to power the LED&#039;s</td></tr><tr><td>horizontal electrophoresis chamber</td><td>Biorad</td><td>1704486EDU</td><td>used to make and run agarose gel in integrity assay</td></tr><tr><td>vertical electrophoresis kit</td><td>Biorad</td><td>1658002EDU</td><td>used to run polyacrylamide gels in calibration curves</td></tr><tr><td>power supply</td><td>Biorad</td><td>1645050&amp;nbsp;</td><td>used to power the electrophoresis</td></tr></tbody></table>

dna staining method based on formazan precipitation induced by blue light exposure

Metodi di colorazione del DNA sono molto importanti per la ricerca biomedica. Abbiamo progettato un metodo semplice che permette la visualizzazione di DNA ad occhio nudo dalla formazione di un precipitato colorato. Funziona immergendo l'acrilamide o gel dell'agarosi del DNA in una soluzione di 1 x (equivalente a 2,0 µM) SYBR Green ho (SG ho) e 0,20 mM blu nitro tetrazolium che produce un viola precipitare di formazan quando esposto alla luce solare o in particolare la luce blu. Inoltre, DNA test di recupero sono state effettuate utilizzando un plasmide ampicillina resistente in un gel di agarosio colorati con il nostro metodo. Un maggior numero di colonie è stato ottenuto con il nostro metodo che con tradizionale che macchia usando SG I con illuminazione ultravioletta. Il metodo descritto è veloce, specifici e non tossico per la rilevazione del DNA, permettendo la visualizzazione di biomolecole ad "occhio nudo" senza un transilluminatore ed è poco costoso e adatto per uso sul campo. Per questi motivi, il nostro nuovo DNA che macchia il metodo ha potenziali benefici all'industria e ricerca.

Un precipitato di formazan viola appare in cui si trova il DNA (verificato da spettrometria di massa13). Nell'esperimento, una scaletta del DNA è stata caricata per fare una curva di calibrazione (Figura 3). Il protocollo funziona per gel di acrilammide sia dell'agarosi, ma ha un'intensità più bassa e richiede più tempo in agarosio. Tuttavia, l'uso del gel dell'agarosi permette il recupero del campione utilizzando un kit disponibil...

Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

Questo metodo permette la colorazione selettiva e quantificazione del DNA in gel immergendo il gel in un SYBR Green mi / Nitro blu di tetrazolio soluzione e poi esporla alla luce solare o una fonte di luce blu. Questo produce un precipitato visibile e quasi non richiede un equipaggiamento, che lo rende ideale per uso sul campo.

DNA che macchia il metodo basato su Formazan precipitazione indotta dall'esposizione alla luce blu

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

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Biochemistry

9.4K Views.  Universidad de Chile. Questo metodo permette la colorazione selettiva e quantificazione del DNA in gel immergendo il gel in un SYBR Green mi / Nitro blu di tetrazolio soluzione e poi esporla alla luce solare o una fonte di luce blu. Questo produce un precipitato visibile e quasi non richiede un equipaggiamento, che lo rende ideale per uso sul campo.

Video: DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

Elettroforesi di DNA usando Thiazole Orange invece di bromuro di etidio o coloranti alternativi

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

Ricerca

Biochimica

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Microscopia a fluorescenza metodi per determinare la vitalità di batteri in associazione con cellule di mammifero

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Immunologia e infezioni

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Alta Tecnica Throughput fluorometrica per la valutazione dei macrofagi fagocitosi e polimerizzazione

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Un sistema integrato di attivare remotamente la trasduzione del segnale intracellulare di fotoattivazione Upconversion Nanoparticle-mediata della chinasi

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Bioingegneria

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Una piattaforma di test anti-biofilm Adatto per l&#39;esplorazione del Biblioteche composto naturale

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.

Here, we present a MATLAB implementation of automated detection and quantitative description of lipid droplets in fluorescence microscopy images of fission and budding yeast cells.

Analisi del contenuto delle goccioline lipidi in Fissione e lieviti in forati utilizzando l'elaborazione automatizzata delle immagini

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Inattivazione di agenti patogeni tramite fotolisi in luce visibile della riboflavina-5′-fosfato

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Visualizzazione della resistenza batterica utilizzando sonde antibiotici fluorescenti

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Vivere la microscopia di fluorescenza delle cellule per osservare i processi essenziali durante la crescita delle cellule microbiche

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Biologia

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Etichettatura specifica dei nucleoidi mitocondriali per la microscopia dell'illuminazione strutturata time-lapse

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...