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

Diese Arbeit wurde unterstützt von Fondo Nacional de Desarrollo y Científico Tecnológico (Fondecyt), Chile, Projekt 11130263 (für CW), Projekt CONICYT ++ NERC Programa de Überwachung Internacional PCI-PII20150073 (für CW) und U-Inicia aus der Vicerrectoría-de Investigación Universidad de Chile (CW).
Danke an Tatiana Naranjo-Palma für die Hilfe in der Anfangsphase des Projekts, Dr. Jorge Babul und Diego Quiroga-Roger für hilfreiche Diskussion, Robert Lesch für die Überarbeitung des Manuskripts und Dirección de Extensión De La spielte de Ciencias Químicas y Farmacéuticas von Universidad de Chile. Vielen Dank auch an Marcelo Pérez für die Videobearbeitung und Neil Stevens zum Korrigieren von Text und Errol Watson für die Bereitstellung von der Voice-over.

1. Vorbereitung und Ausführung der Gel
<ol><li>Bereiten Sie die nicht Denaturierung 12 % Polyacrylamid-Gel Gele Sodium Dodecyl Sulfat-Polyacrylamid-Gel-Elektrophorese (SDS-PAGE) Rezept fand in Sambrook und Russell14 , aber mit Wasser anstelle von SDS.<ol>
<li>Verwenden Sie um 5 mL Lösung Gel vorzubereiten, 1,7 mL destilliertem Wasser, 2 mL Acrylamid/Bis-Acrylamid (30/0,8 % w/V), 1,3 mL 1,5 M Tris pH 8,8, 0,05 mL 10 % Ammonium bleichen und 0,002 mL TEMED.</li>
		<li>Zur Vorbereitung verwenden 2 mL Stapeln Gel 1,5 mL Wasser 0,33 mL Acrylamid/Bis-Acrylamid (30/0,8 % w/V), 0,25 mL 1 M Tris pH 6.8, 0,02 mL 10 % Ammonium bleichen und 0,002 mL TEMED. Die Abmessungen des Gels sind 7,3 x 8,2 cm x 0,075 cm (l x w x d). Hinweis: Verwenden Sie einen 15 gut Kamm von 0,75 mm Dicke für die optimale Empfindlichkeit.</li>
	</ol>
</li>
	<li>Fügen Sie die entsprechenden Beträge von 6 x laden Puffer und DNA-Probe zu einem 1,5 mL Microcentrifuge Schlauch (z.B. Verwendung 1 µL 6 X laden Puffer 5 µL DNA-Probe).</li>
	<li>Legen Sie das Gel in der vertikalen Elektrophorese-Kammer.<ol>
<li>Füllen Sie die Kammer mit 750 mL 1 X TAE Puffer 2 Gele ausgeführt. 1 L 50 vorbereiten X TAE, 442 g Tris-Base, 57,1 mL Eisessig und 100 mL 0,5 M EDTA pH 8 zu verwenden. Fügen Sie destilliertes Wasser hinzu, um ein finales Volumen von 1 L.</li>
		<li>Laden Sie die Proben in das Gel. Führen Sie das Gel bei 100 V 2 h.</li>
	</ol>
</li>
	<li>Alternativ bereiten Sie eine 1 X TAE Agarose-Gel Rezept gefunden in Sambrook14.<ol>
<li>Geben Sie 300 mL 1 X TAE der horizontalen Elektrophorese-Kammer. Führen Sie das Gel für 45 min bei 100 V.</li>
	</ol>
</li>
</ol>(2) NBT-SG ich Färbung (Abbildung 1).
<ol><li>
Acrylamid-Gel Färbung.
	<ol>
<li>Bereiten Sie die Lösungen: 0,1 % w/V NBT und 1,2 x SG ich. Hinweis: Es ist möglich, verwenden Sie Interkalation Bromid, GelRed und SYBR Gold statt SG ich, aber ich hatte die beste Leistung SG.</li>
		<li>Gießen Sie 16,75 mL 1.2 X SYBR grün I Lösung in ein Gefäß passender Größe. Hinweis: Wir den Deckel einer Pipette-Tipp-Box als Container verwendet, und diese Bände werden berechnet, um das Gel mit diesem Container abzudecken; seine Abmessungen sind 11,6 x 7,6 cm x 2,6 cm (l x w x d).</li>
		<li>Legen Sie das Gel in die Lösung. Hinzufügen von 3,25 mL 0,1 % w/V NBT-Lösung 0,2 mM NBT geben und 1 X (entspricht 2,0 µM) SG ich Endkonzentrationen.</li>
		<li>Verschließen Sie den Behälter mit Kunststoff-Folie, und wickeln Sie komplett den Behälter mit Aluminiumfolie, Umgebungslicht zu blockieren. Hinweis: Die Kunststoff-Folie wird verwendet, um Spritzer zu vermeiden und um Reaktion der Aluminiumfolie mit der Färbelösung zu vermeiden.</li>
		<li>25 min auf einem Orbitalschüttler bei 60 u/min schütteln.</li>
		<li>Entfernen Sie Aluminium und Kunststoff-Abdeckung. Für 90 min. maximale Sonnenbestrahlung aussetzen. Überprüfen Sie alle 5 Minuten, bis die Band von Interesse erscheint.</li>
		<li>Um blaues Licht zu verwenden, bereiten Sie eine Lochrasterplatinen mit 3 Leuchtdioden (LED) und legen Sie die LEDs ca. 5 mm von der Geloberfläche (überprüfen Sie die Färbung kontinuierlich, wie die Zeit von der Intensität der LED-Lichtquelle abhängen wird benötigt).</li>
	</ol>
</li>
	<li>
Agarosegel Färbung.
	<ol>
<li>Nach der Vorbereitung und Ausführung einer 1 % Agarose gel wie in den Abschnitten 1.4 beschrieben (die Maße sind 1 x 6,2 cm x 7 cm; l x w x t), die gleichen Schritte wie in 2.1 mit diesen Änderungen:</li>
		<li>Gießen Sie 41,9 mL 1,2 x SG ich Lösung in den Deckel einer Pipette-Tipp-Box; seine Abmessungen sind 11,6 x 7,6 cm x 2,6 cm (l x w x d). 8,1 mL 0,1 % NBT-Lösung zugeben.</li>
		<li>Verschließen Sie den Behälter mit Kunststoff-Folie, und dann wickeln Sie vollständig den Behälter mit Aluminiumfolie, Umgebungslicht zu blockieren.</li>
		<li>1 h auf einem Orbitalschüttler bei 60 u/min schütteln.</li>
	</ol>
</li>
</ol>(3) Datenanalyse.
<ol><li>
NBT-SG ich Kalibrierkurven.
	<ol>
<li>Gelbilder durch Scannen bei 1200 dpi zu erhalten.</li>
		<li>Offene Software für die Bildanalyse. Die Bilder mit einem Bildbearbeitungsprogramm zu begradigen. Führen Sie eine Bild-Densitometrie.</li>
		<li>Berechnen Sie die Fläche unter der Kurve. Bereiten Sie ein Diagramm der Signal/SignalMax im Vergleich zu DNA-Konzentration.</li>
	</ol>
</li>
</ol>4. DNA-Recovery.
<ol><li>Bereiten Sie eine 1 % Agarose-Gel in 1 X TAE mit dem Rezept in Sambrook14gefunden.</li>
	<li>Geben Sie 300 mL 1 X TAE der horizontalen Elektrophorese-Kammer. Laden Sie 4 µL von 100 ng/µL pDJ10015 Plasmid DNA-Leiter nach dem Schema in Abbildung 2angegeben. Führen Sie das Gel für 1 h bei 100 V.</li>
	<li>Markieren Sie und wiegen Sie zwei 2 mL Mikrozentrifugenröhrchen.</li>
	<li>Schneiden Sie das Gel in der Mitte um zwei identische Hälften haben. Jeder sollte die Leiter und das Probe-Plasmid, vergleichen Sie die verschiedenen befleckenden Techniken enthalten. Eine schematische Darstellung ist in Abbildung 2.</li>
	<li>Flecken auf einer Hälfte mit NBT-SG I wie in Schritt 2.<ol>
<li>Die Plasmid-Band mit einem sauberen Skalpell ausgeschnitten. Speichern Sie das Gel-Stück in eine Röhre und wiegen.</li>
	</ol>
</li>
	<li>Fleck, den andere Teil des Gels mit SG ich mit 50 mL 1 x SG ich Lösung und langsam schütteln (60 u/min) das Gel für 1 h in der Dunkelheit.<ol>
<li>Legen Sie das Gel auf ein Transilluminator und für 2 min. Schnitt aus dem Plasmid-Band mit einem sauberen Skalpell aussetzen.</li>
		<li>Speichern Sie das Gel Stück in die zweite Röhre und wiegen. Hinweis: Das Protokoll kann hier angehalten werden, wobei die Rohre bei-20 ° C.</li>
	</ol>
</li>
	<li>Plasmid Extraktion mithilfe eines kommerziellen Gel Extraction Kits führen.</li>
</ol>(5) Plasmid Normalisierung.
<ol><li>Fügen Sie in einem 1,5 mL Microcentrifuge Schlauch 10 µL des extrahierten Plasmid und 60 µL des Typs, den ich Wasser hinzu. Mix und übertragen auf eine 60 µL-Quarz-Küvetten oder 2 µL direkt in eine Nanodrop verwenden.</li>
	<li>Messen Absorptionsspektren zwischen 230 und 320 nm. Berechnen Sie das Beispiel Plasmid Konzentration anhand der folgenden Formel: <img alt="Equation 1" src="/files/ftp_upload/56528/56528eq1v3.jpg"> wo D ist der Verdünnungsfaktor angewendet (in diesem Fall ist es 70/10), AX ist die Absorption bei x nm und 50 ist der Umrechnungsfaktor µg/ml für DsDNA.</li>
	<li>Verdünnte Proben auf 9,1 ng/µL.</li>
	<li>Escherichia coli (E. Coli) kompetente Zellen (z. B. XL10 Gold) zu verwandeln nach der Inoue Transformation Verfahren16 mit 5 µL der verdünnten Probe.</li>
	<li>Die Anzahl der Kolonien in den Petrischalen und beachten Sie die Verdünnung. Berechnen Sie die Kolonie Bildenden Einheiten (KBE) mit der folgenden Formel17: <img alt="Equation 2" src="/files/ftp_upload/56528/56528eq2v2.jpg"> wo D ist der Verdünnungsfaktor von der ursprünglichen Kultur angewendet.</li>
	<li>Führen Sie einen ungepaarten t-Test.</li>
</ol>

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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. 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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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Eine sensible und Roman DNA Färbung Methode anhand der Reduzierung der NBT und die Verwendung von SG, die ich in diesem Artikel vorgestellt wurde. Der entscheidende Schritt in diesem Protokoll ist die Inkubationszeit des Gels in der NBT-SG ich Lösung und die Konzentration der NBT. Die Färbung Agarose-Gele dauert länger als bei Acrylamid Gele wegen der größeren Dicke Agarose-Gele. Diese Methode funktioniert nicht gut im Beisein von SDS, wie NBT mit ihm in Berührung ausfällt. Wenn das Gel einen violetten Hintergrun...

Mit den Fortschritten in der Biochemie und Molekularbiologie haben die Studien mit DNA bessere Techniken zur DNA Analyse erforderlich. Die erste Methode für DNA-Färbung wurde Silber Färbung, die sehr empfindlich ist, aber ermangelt Selektivität und erlaubt keine Probenrückgewinnung. Später, erlaubt die Entwicklung von fluoreszierenden DNA-Färbung die selektive Quantifizierung der DNA mit der Möglichkeit der Probenrückgewinnung. Eines der ersten Fluoreszenzfarbstoffen verwendet für DNA-Quantifizierung war Interkalation Bromid1, mutagene2. Allerdings gibt es nun verbesserte fluoreszierende Farbstoffe, die sicherer und empfindlicher, wie GelRed und SYBR Green sind ich (SG ich)3; aber alle diese Leuchtstofffärbungen erfordern den Einsatz eines ultravioletten (UV) Transilluminator oder Fluorimeter.
Es gibt andere Techniken zum Färben sichtbar DNA, wie Methylenblau4 und Kristallviolett5,6,7,8,9, aber all diese Leiden verringerte Empfindlichkeit und Selektivität. Die Tetrazolium-Salze sind organische Verbindungen, die anfällig für Reduktion, und in diesem Fall bilden sie eine unlösliche und farbigen Formazan überstürzen sich10,11. Vor kurzem zeigten einige bivalenten Tetrazoliumsalz Salze aufgrund ihrer positiven Tetrazoliumsalz Ringe12an DNA zu binden.
In den letzten Veröffentlichung13wurde eine neue sichtbare Technik zu färben und zu quantifizieren DNA in Polyacrylamid-Gele vorgeschlagen, mit der Reduktion von bivalenten Tetrazoliumsalz Salz namens Nitro blau Tetrazoliumsalz (NBT) und Fluoreszenz-Farbstoffen wie Interkalation Bromid, GelRed, SYBR Green I und SYBR Gold. Diese Reaktion bei Anwesenheit von blauem Licht oder Sonnenlicht gearbeitet und erlaubt Probenrückgewinnung mit verbesserter Qualität gegenüber dem Einsatz von SG ich mit einem UV-Transilluminator. Das Ziel dieses Papiers ist ein detailliertes Protokoll der Färbung Technik mit Tetrazoliumsalz bieten Salze.

<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

DNA-Färbung Methoden sind sehr wichtig für die biomedizinische Forschung. Wir haben eine einfache Methode, die DNA-Visualisierung mit dem bloßen Auge durch die Bildung von farbigen Niederschlag ermöglicht entwickelt. Es funktioniert durch Einweichen der Acrylamid oder DNA Agarose-gel in einer Lösung aus 1 X (entspricht 2,0 µM) SYBR Green ich (SG ich) und 0,20 mM Nitro blau Tetrazoliumsalz, die eine lila produziert Niederschlag von Formazan bei Sonneneinstrahlung oder speziell blaues Licht. Auch wurden DNA-Recovery Tests durchgeführt mit einem Ampicillin resistent Plasmid in einem Agarosegel mit unserer Methode befleckt. Eine größere Zahl von Kolonien wurde erreicht mit unserer Methode als mit traditionellen Färbung mit SG ich mit UV-Beleuchtung. Das beschriebene Verfahren ist schnell, spezifisch und nicht toxisch für DNA-Nachweis, so dass Visualisierung von Biomolekülen mit dem "bloßen Auge" ohne ein Transilluminator und ist billig und für den mobilen Einsatz geeignet. Aus diesen Gründen hat unsere neue DNA Färbung Methode potenzielle Nutzen für Forschung und Industrie.

Ein violette Formazan Niederschlag erscheint (verifiziert durch Massenspektrometrie13) wo die DNA befindet. Im Experiment wurde eine DNA-Leiter geladen zu einer Kalibrierkurve (Abbildung 3). Das Protokoll arbeitet für Acrylamid und Agarose Gele, aber es hat eine geringere Intensität und nimmt eine längere Zeit in Agarose. Jedoch die Verwendung von Agarose-Gele ermöglicht die Rückgewinnung der Probe mit einem kommerziell erhältlic...

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

Diese Methode erlaubt selektive Färbung und Quantifizierung der DNA in den Gelen durch Einweichen das Gel in einem SYBR Grün ich / Nitro blau Tetrazoliumsalz Lösung und setzen Sie sie dem Sonnenlicht oder eine blaue Lichtquelle. Dies erzeugt einen sichtbaren Niederschlag und erfordert fast keine Ausrüstung, wodurch es ideal für den mobilen Einsatz.

DNA-Färbung Methode basiert auf Formazan Niederschlag induziert durch blaues Licht aussetzen

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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JoVE Journal

Biochemistry

9.4K Aufrufe.  Universidad de Chile. Diese Methode erlaubt selektive Färbung und Quantifizierung der DNA in den Gelen durch Einweichen das Gel in einem SYBR Grün ich / Nitro blau Tetrazoliumsalz Lösung und setzen Sie sie dem Sonnenlicht oder eine blaue Lichtquelle. Dies erzeugt einen sichtbaren Niederschlag und erfordert fast keine Ausrüstung, wodurch es ideal für den mobilen Einsatz.

Serie: 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.

DNA-Elektrophorese mit Thiazole Orange statt Interkalation Bromid oder Alternative Farbstoffe

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

Forschung

Biochemie

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.

Fluoreszenz-Mikroskopie-Methoden zur Bestimmung der Lebensfähigkeit der Bakterien in Verbindung mit Säugerzellen

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

Immunologie und Infektion

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.

High Throughput Fluorometric Technik zur Bewertung der Makrophagen-Phagozytose und Aktinpolymerisation

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.

Ein integriertes System aus der Ferne intrazelluläre Signaltransduktion von Großbildschirmen Nanoparticle vermittelte Kinase Photoaktivierungen auslösen

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

Biotechnik

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.

Eine Plattform von Assays Anti-Biofilm auf die Erforschung der natürlichen Substanzbibliotheken Geeignet

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.

Analyse des Lipidtröpfchengehalts in Spalt- und Budding-Hefen mittels automatisierter Bildverarbeitung

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.

Inaktivierung von Krankheitserregern durch Photolyse von Riboflavin-5′-Phosphat im sichtbaren Licht

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.

Visualisierung der bakteriellen Resistenz mit fluoreszierenden Antibiotikasonden

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.

Live Cell Fluoreszenzmikroskopie um wesentliche Vorgänge während der mikrobiellen Zellwachstum beobachten

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

Biologie

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.

Spezifische Kennzeichnung von mitochondrialen Nukleoiden für die Zeitraffer-Struktur-Beleuchtungsmikroskopie

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