Diese Arbeit wurde von der National Science Foundation Environmental Genomics Program (Grant No EF-0723707) gefördert.

1. Microbial Cell Extraction: <ol><li> Homogenisieren mikrobiellen Matten mit einer sterilen Schleifen Pistill durch Mischen gründlich. Legen Sie etwa alle 30 g (Feuchtgewicht) homogenisiert Matte Material in sterilen Behälter von Waring, etwa 100 ml 1 M NaCl (oder eine Konzentration spezifischen Probe in use) hinzufügen, und fügen sich dreimal bei mittlerer Geschwindigkeit für 1 min mit intermittierende Kühlung in einem -20 ° C Gefrierschrank für 1 min. Übertragen Sie die Gülle in einen 250 mL Zentrifuge Flasche füllen und die restlichen leeren Volumen mit 1 M NaCl. Bereiten NaCl-Lösung in autoklaviert DI Wasser und Filter sterilisieren. </li><li> Weitere verdrängen die mikrobielle Zellen aus der Matrix durch Schütteln (150 rpm) mit einem Vortexer (Vortex-Genie R 2) bei Raumtemperatur für 30 min. </li><li> Zentrifugation bei niedriger Geschwindigkeit (500x g) für 15 min bei 4 ° C. Vorsichtig den Überstand in einen Kolben mit minimaler Störung des Sediments. </li><li> Mit dem pelletiert Sediment, wiederholen Sie die Schritte Mischen und 1,2 und 1,3 weitere vier Male mit der Zugabe von frischem NaCl jedem Sediment Pellet am Ende von Schritt 1.3. Der Überstand aus jeder NaCl-Extraktion ist in einem frischen Kolben überführt. </li><li> Kombinieren Sie die Überstände aus den 5 Zellen Extraktionen und Zentrifuge in Aliquots von 200 ml mit hoher Geschwindigkeit (25.000 xg) für 15 min bei 4 ° C. Überstand verwerfen, resuspendieren jedem Zellpellet in 10 ml 2% Natriumhexametaphosphat, kombinieren Pellets und machen auf ein Volumen von 200 ml mit 2% Natriumhexametaphosphat. Wash-Zellen durch Schütteln (150 rpm) bei Raumtemperatur für 30 min. Am Ende dieses Schrittes sollten Sie nur eine Röhre von Zellen, um zum nächsten Schritt übergehen. </li><li> Zentrifuge mit hoher Geschwindigkeit (25.000 xg) für 15 min bei 4 ° C. Überstand verwerfen, Zellpellet resuspendieren in 200 ml TE (50 mM EDTA), und waschen Sie die Zellen durch Schütteln (150 rpm) bei Raumtemperatur für 10 min. </li><li> Zentrifuge mit hoher Geschwindigkeit (25.000 xg) für 15 min bei 4 ° C, den Überstand verwerfen, in 15 mL TE (10 mM EDTA) resuspendiert, und speichern 200-ul Aliquots bei -80 ° C bis zum Gebrauch für die DNA-Extraktion. </li></ol> 2. DNA Extraktion und Reinigung: <ol><li> Geben Sie 200 ul 5 M NaCl, 200 ul 10% SDS und 100 ul 14,3 Mio. β-Mercaptoethanol auf die 200-ul mikrobielle Zelle Aliquot. Vorsichtig mischen durch Invertieren 4-6 mal. </li><li> Vorbehaltlich der Mischung auf 3 Runden Frost-Tau durch Eintauchen in flüssigen Stickstoff für 2 min durch Auftauen in einem 65 ° C Wasserbad für 5 min. Extend endgültigen Auftauen bis 10 min. </li><li> Geben Sie 200 ul 5 M Kaliumacetat (pH 5,5) und auf Eis für 10 min. Zentrifuge bei 10.000 xg für 10 min bei 4 ° C und den Überstand in ein neues Röhrchen mit einem weit Bohrung Pipettenspitze. Add 3 ul RNase A und Inkubation bei 37 ° C für 1 Stunde. </li><li> Add gleichen Volumen Chloroform, kurz schütteln durch Umdrehen und Zentrifuge bei 15.000 xg für 10 min bei Raumtemperatur. </li><li> Sorgfältig Transfer die oberste Schicht der Überstand in ein neues Röhrchen mit einem weit Bohrung Pipettenspitze. Vermeiden Pipettieren der weiße Grenzschicht zwischen der oberen und unteren Schichten entfernt. Wiederholen Sie die Chloroform-Extraktion. </li><li> Fügen Sie dem gleichen Volumen gekühlt (bei -20 ° C) Isopropanol zum Überstand und inkubieren auf Eis für 30 min zur Fällung DNA. </li><li> Zentrifuge bei Höchstgeschwindigkeit (20.800 xg) für 10 min bei 4 ° C bis Pellet DNA. </li><li> Überstand verwerfen und waschen DNA-Pellet mit 1 ml gekühlt (-20 ° C) 70% Ethanol. </li><li> Zentrifuge bei Höchstgeschwindigkeit (20.800 xg) für 10 min bei 4 ° C, den Überstand verwerfen, wiederholen Sie 70% Ethanol waschen und an der Luft trocknen DNA-Pellet für 10 min. Wiederholen Sie die 70% Ethanol waschen. </li><li> Resuspendieren DNA in 25 ul TE (10 mM EDTA), warm bei 65 ° C für 5 min in einer Röhre zu verbinden, wenn mehrere Rohre verwendet wurden, und Make-up-Volumen auf 500 ul mit TE. </li><li> Add 500 mL eiskaltem (oder bei 4 ° C) 20% Polyethylenglykol (PEG) (hergestellt in 1,2 M NaCl). Vorsichtig mischen durch Umdrehen, auf Eis für 10 min und Zentrifuge inkubieren bei maximaler Geschwindigkeit für 10 min bei 4 ° C bis Pellet DNA. Entfernen und den Überstand verwerfen und waschen Sie die pelletierte DNA mit 1 ml gekühltem 70% Ethanol, an der Luft trocknen für 10 min und Resuspendieren in 30-50 ul TE oder molekulare reines Wasser. Erleichterung Resuspension der DNA durch Erwärmung auf 65 ° C für 5 min und DNA bei -80 ° C. </li></ol> 3. DNA-Reinheit, Konzentration und Größe Bestimmung: <ol><li> Bestimmen DNA-Qualität durch die Messung des A 260/280 und A 260/230 Verhältnisse mit einem Nanodrop 1000 Spektralphotometer oder sonstige geeignete Instrumente. </li><li> Bestimmen DNA-Konzentration mit dem Quant-iT dsDNA Assay Kit nach den Anweisungen des Herstellers. </li><li> Bestimmen DNA Größe mit Puls-Feld-Gelelektrophorese. Bereiten Sie 1% Agarosegel mit 0,5 X TBE und verwenden Sie den folgenden Parameter auf der CHEF Mapper XA System; ersten Einschalten der Zeit von 0,35 s, eine endgültige Zeit umschalten von 7,67 s und 120 ° Öffnungswinkel für ein Gefälle von 6,0 V / cm bei eine lineare rAmping-Faktor. Stain Gel mit 1 x SYBR Green I für 30 min. Legen Sie zwischen 700-1000 ng Gesamt-DNA auf einem Gel aufgetrennt. Siehe Abb.. 3 für eine verkürzte Form der komplette Protokoll. </li></ol> Repräsentative Ergebnisse: Zell-Extraktion: Sequential mikrobiellen Zellextrakten (Überstände) haben gezeigt, dass die Trübung der Extrakte als die Zahl der Extraktionen erhöhen abnimmt. Dies deutet auf eine Reduzierung der Anzahl der Zellen nach jeder aufeinander folgenden Zelle Extraktion. Wichtig hierbei ist, dass da jeder zusätzliche Zelle Extraktionsschritt bietet die Möglichkeit für Kontaminanten Einführung, die Anzahl der Zellen Extraktionen minimiert werden sollte, so dass die endgültige Zellpellet repräsentativ für die Gesamtheit der mikrobiellen Gemeinschaft ist. Andere Quellen von Verunreinigungen wurden durch die Verabschiedung angemessener Labor sterile Techniken gesteuert. Zum Beispiel wurden die Lösungen in autoklaviert DI Wasser hergestellt und sterilisiert. Die Behälter wurden mit Alkohol sterilisiert, autoklaviert und behandelt unter UV-Licht und UV-Vernetzer. DNA-Konzentration und Qualität Bestimmung: Das Protokoll ergab etwa 2 ug HMW-DNA (35-50 kb) (Abb. 4) pro Gramm Matte Probe mit einem A 260/280 Ratio von 1,6 und ein A 260/230 Ratio von 0,7 (Tabelle 1). Obwohl die A 260/230 Ratio schien gering zu sein, keine Hemmung der nachgeschalteten molekularen-basierten Anwendungen wie PCR-Amplifikation des 16S rRNA-Gene in einer separaten Studie 7 beobachtet wurde. Es ist wichtig, dass DNA aus hypersaline Matten hat zwei Hauptquellen der Kontamination beachten Sie, EPS und Salze. Es ist daher möglich, dass Spuren dieser Verunreinigungen kann Einfluss auf die A 260 / 230 Verhältnisse trotz der enormen Anstrengungen, um ihre Auswirkungen auf die Downstream-Anwendungen zu reduzieren. DNA Größenbestimmung: Pulse-Feld-Gel-Elektrophorese verwendet eine pulsierende Stromfluss mit Intervallschaltung direktionale Schalter was zu einem DNA-Abstrich, wie in Abbildung 3 dargestellt. Unser Protokoll ergab einen HMW-DNA von ungefähr 35-50 kb. Während einige DNA Größe kann kleiner sein als 30 kb, ist es wichtig, einen Teil der Abstrich über 35 kb da Fosmid Klonen ~ 40 kb DNA-Inserts erfordert und größere DNA-Fragmente bieten einen besseren Zugang zu erhalten Biosynthesewege haben. In unseren Untersuchungen wurde die DNA-Abstrich über 35 kb ausgeschnitten und gereinigt für große Insert-Vektor Klonierung und andere molekulare Anwendungen. <img src="/files/ftp_upload/2887/2887fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Salzwasserwanne mikrobiellen Matte Probenahmestelle (Big Pond) auf Eleuthera, Bahamas entfernt. <img src="/files/ftp_upload/2887/2887fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Ein Querschnitt der hypersaline mikrobiellen Matte in dieser Studie verwendet. Die mikrobielle Matte wurde aus einer hypersaline Teich auf Eleuthera, Bahamas befindet sich erhalten. <img src="/files/ftp_upload/2887/2887fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Schematische Darstellung der Verfahren in mikrobielle Zelle Extraktion, Zell-Lyse, Extraktion und Reinigung von DNA metagenomische beteiligt. <a href="http://www.jove.com/files/ftp_upload/2887/2887fig3large.jpg">Klicken Sie hier, um ein größeres Bild zu sehen.</a> <img src="/files/ftp_upload/2887/2887fig4.jpg" alt="Abbildung 4"/> Abbildung 4. Molekulargewicht Charakterisierung der extrahierten DNA mittels Puls-Feld-Gelelektrophorese. Lane M ist ein HMW-Marker und die Spuren 1 und 2 sind Wiederholungen von Metagenom DNA aus dem Eleuthera hypersaline Matte mit dem obigen Protokoll extrahiert. <table border="1"><tbody><tr><td> Extraction Method </td><td></td><td> Konzentration ng / g </td><td> A 260/280 </td><td> A 260/230 </td></tr><tr><td> PEG </td><td> REP 1 </td><td> 1806,1 </td><td> 1,64 </td><td> 0,76 </td></tr><tr><td></td><td> Rep 2 </td><td> 2010,8 </td><td> 1,61 </td><td> 0,75 </td></tr></tbody></table> Tabelle 1. Die Messung der Konzentration und Qualität der DNA extrahiert aus mikrobiellen hypersaline Matte.

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	<li>Decho, 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=Microbial+biofilms+in+intertidal+systems:+an+overview.">Microbial biofilms in intertidal systems: an overview.</a> Cont. Shelf Res. 20, 1257-1273 (2000).</li><li>Dupraz, C., Visscher, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+lithification+in+marine+stromatolites+and+hypersaline+mats.">Microbial lithification in marine stromatolites and hypersaline mats.</a> Trends Microbiol. 13, 429-438 (2005).</li><li>Steffan, R. J., Goksoyr, J., Boj, A. K., Atlas, R. M. <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+soils+and+sediments.">Recovery of DNA from soils and sediments.</a> Appl. Environ. Microbiol. 54, 2908-2915 (1988).</li><li>Lee, Y. K., Kim, H. W., Liu, C. L., Lee, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+DNA+extraction+from+marine+bacteria+that+produce+extracellular+materials.">A simple method for DNA extraction from marine bacteria that produce extracellular materials.</a> J. Microbiol. Methods. 52, 245-250 (2003).</li><li>Roose-Amsaleg, C. L., Garnier-Sillam, E., Harry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+Purification+of+Microbial+DNA+from+Soil+and+Sediment+Samples.">Extraction and Purification of Microbial DNA from Soil and Sediment Samples.</a> Appl. Soil Ecol. 18, 47-60 (2001).</li><li>de Lipthay, J. R., Enzinger, C., Johnsen, K., Aamand, J., S&#248;rensen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+DNA+extraction+method+on+bacterial+community+composition+measured+by+denaturing+gradient+gel+electrophoresis.">Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis.</a> Soil Biol. Biochem. 36, 1607-1614 (2004).</li><li>Bey, B. S., Fichot, E. B., Dayama, G., Decho, A. W., Norman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+High+Molecular+Weight+DNA+from+Microbial+Mats.">Extraction of High Molecular Weight DNA from Microbial Mats.</a> Biotechniques. 49, 631-640 (2010).</li><li>Kakirde, S. K., Parsley, L. C., Liles, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+does+matter:+Application-driven+approaches+for+soil+metagenomics.">Size does matter: Application-driven approaches for soil metagenomics.</a> Soil Biology &#38; Biochemistry. 42, 1911-1923 (2010).</li><li>Rodon, M. R., August, P. R., Bettermann, A. D., Brady, S. F., Grossman, T. H., Liles, M. R., Loiacono, K. A., Lynch, B. A., MacNeil, I. A., Minor, C., Tiong, C. L., Gilman, M., Osburne, M. S., Clardy, J., Handelsman, J., Goodman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+the+Soil+Metagenomic:+A+Strategy+for+Accessing+the+Genetic+and+Functional+Diversity+of+Uncultured+Microorganisms.">Cloning the Soil Metagenomic: A Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms.</a> Appl. Environ. Microbiol. 66, 2541-2547 (2000).</li><li>Beja, O., Aravind, L., Koonin, E. V., Suzuki, M. T., Hadd, A., Nguyen, L. P., Jovanovich, S. B., Gates, C. M., Feldman, R. A., Spudich, J. L., Spudich, E. N., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Rhodopsin:+Evidence+for+a+New+Type+of+Phototrophy+in+the+Sea.">Bacterial Rhodopsin: Evidence for a New Type of Phototrophy in the Sea.</a> Science. 289, 1902-1906 (2000).</li></ol>

Da die gesamte Zelle Entnahme aus komplexen und sehr unterschiedlichen mikrobiellen Matte Proben nicht praktikabel ist, ist die wichtigste Angelegenheit, wie gut die extrahierten Zellen die gesamte mikrobielle Matte Gemeinschaft darstellen. In einer früheren Studie zeigte PCR-DGGE Analyse mikrobieller 16S rRNA-Gene, dass die fünf Zelle Ausbauschritte in diesem Protokoll verwendeten Zellen, die repräsentativ für die Gesamtheit mikrobiellen Matte Gemeinschaft 7 sind Extrakte. Die tatsächliche Anzahl der Ze...

<table border="1"><tbody><tr><th> Name des Reagenz </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> β-Mercaptoethanol </td><td> Sigma-Aldrich </td><td> M3148 </td><td></td></tr><tr><td> Polyethylenglycol 8000 </td><td> Promega </td><td> V3011 </td><td> 20% in 1,2 M NaCl </td></tr><tr><td> Kaliumacetat </td><td> Fisher Scientific </td><td></td><td> Fisher Scientific </td></tr><tr><td> Quant-iT dsDNA Assay Kit </td><td> Invitrogen </td><td> Q33130 </td><td></td></tr><tr><td> RNase </td><td> Epicentre </td><td> MRNA092 </td><td></td></tr><tr><td> Kochsalz </td><td> BDH Chemicals </td><td> BDH8014 </td><td> Entsprechende konz. </td></tr><tr><td> Natriumdodecylsulfat </td><td> Fisher Scientific </td><td> 03-500-509 </td><td> 10% in Wasser </td></tr><tr><td> Natriumhexametaphosphat </td><td> EMD Chemicals </td><td> SX0583-3 </td><td> 2% in Wasser </td></tr><tr><td> TBE </td><td> Fisher Scientific </td><td> BP1333-1 </td><td></td></tr><tr><td> CHEF Mapper XA-System </td><td> Bio-Rad Laboratories </td><td> 170-3670 </td><td></td></tr><tr><td> NanoDrop 1000 Spektralphotometer </td><td> Thermo Scientific </td><td> ND-1000 </td><td></td></tr><tr><td> Vortexer </td><td> Scientific Industries Inc. </td><td></td><td></td></tr><tr><td> Ultraviolet Crosslinker </td><td> UVP </td><td></td><td></td></tr><tr><td> Waring </td><td> Waring Labor </td><td> LB10S </td><td></td></tr></tbody></table>

extraction of high molecular weight dna from microbial mats

Erfolgreiche und genaue Analyse und Interpretation von Daten metagenomische ist abhängig von der effizienten Gewinnung von hochwertigen, mit hohem Molekulargewicht (HMW)-Community DNA. Doch Umwelt-mat Proben stellen häufig Schwierigkeiten zu erhalten hohe Konzentrationen von hoher Qualität, HMW-DNA. Hypersalinen mikrobiellen Matten enthalten hohe Mengen an extrazellulären polymeren Substanzen (EPS) 1 und Salze, die Downstream-Anwendungen der extrahierten DNA hemmen können. Direkte und harte Methoden sind oft in der DNA-Extraktion aus feuerfestem Proben verwendet. Diese Methoden sind in der Regel verwendet, da das EPS in Matten, Klebstoff-Matrix, bindet DNA 2,3 während der direkten Lyse. Als Folge der härteren Extraktionsmethoden, DNA wird in kleinen Größen 4,5,6 fragmentiert. Die DNA wird somit ungeeignet für große Insert-Vektor Klonen. Um diese Einschränkungen zu umgehen, melden wir eine verbesserte Methodik zur HMW-DNA von guter Qualität und Quantität von hypersaline mikrobiellen Matten extrahieren. Wir verwendeten eine indirekte Methode, die die Trennung von mikrobiellen Zellen aus dem Hintergrund Matte Matrix durch Mischen und differentielle Zentrifugation. Eine Kombination aus mechanischen und chemischen Verfahren wurde verwendet, um zu extrahieren und zu reinigen DNA aus den extrahierten mikrobieller Zellen. Unser Protokoll ergibt etwa 2 ug HMW-DNA (35-50 kb) pro Gramm Matte Probe, mit einer A 260/280 Ratio von 1,6. Darüber hinaus schlägt Amplifikation von 16S rRNA-Gene 7, die das Protokoll in der Lage, minimieren oder zu eliminieren hemmenden Wirkungen von Verunreinigungen ist. Unsere Ergebnisse liefern eine geeignete Methode zur Extraktion von HMW-DNA aus mikrobiellen Matten für funktionale metagenomische Studien und kann auf andere Umwelt-Proben, aus denen die DNA-Extraktion ist eine Herausforderung.

Watch this Scientific Journal Video about Extraction of High Molecular Weight DNA from Microbial Mats at JoVE.com

Extraction of High Molecular Weight DNA from Microbial Mats

Wir bieten ein verbessertes Protokoll zur Extraktion von DNA mit hohem Molekulargewicht aus hypersaline mikrobielle Matten. Mikrobielle Zellen sind von der Matte Matrix vor der DNA-Extraktion und Reinigung getrennt. Dies erhöht die Konzentration, Qualität und Größe der DNA. Das Protokoll kann für andere feuerfeste Proben verwendet werden.

Extraktion von DNA mit hohem Molekulargewicht von mikrobiellen Matten

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

extraction-of-high-molecular-weight-dna-from-microbial-mats

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Biology

19.4K Aufrufe.  Arnold School of Public Health, University of South Carolina. Wir bieten ein verbessertes Protokoll zur Extraktion von DNA mit hohem Molekulargewicht aus hypersaline mikrobielle Matten. Mikrobielle Zellen sind von der Matte Matrix vor der DNA-Extraktion und Reinigung getrennt. Dies erhöht die Konzentration, Qualität und Größe der DNA. Das Protokoll kann für andere feuerfeste Proben verwendet werden.

Serie: Extraction of High Molecular Weight DNA from Microbial Mats

Biology of Microbial Communities  - Interview

Biologie mikrobieller Gemeinschaften - Interview

Forschung

Biologie

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

DNA Extraction from 0.22 &amp;mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

DNA-Extraktion von 0,22 pM Sterivex Filter und Cäsiumchlorid-Dichtegradientenzentrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Extraktion von High Molecular Weight genomischer DNA aus Böden und Sedimenten

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

DNA-Extraktion aus in Paraffin eingebettete Material für genetische und epigenetische Analysen

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Ein Hybrid-DNA-Extraktionsmethode für die qualitative und quantitative Bewertung der bakteriellen Gemeinschaften aus Zucht Proben

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

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

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

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

Mucin Agarose-Gel-Elektrophorese: Western Blotting für Hochmolekulare Glykoproteine


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

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Herstellung von Formalin-fixierten und in Paraffin eingebetteten Gewebe Cores sowohl für RNA und DNA-Extraktion

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

Modifizierte Methoden zum Laden von DNA-Extraktionsplatten mit hohem Durchsatz reduzieren das Kontaminationspotenzial

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolierung von hochwertigen atrialen und ventrikulären Myozyten der Maus für die simultane Messung von Ca2+ Transienten und L-Typ Calciumstrom

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Kleine Extraktion von Caenorhabditis elegans Genomischer DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...