Questo lavoro è stato finanziato dal Programma National Science Foundation Environmental Genomica (Grant No. EF-0723707).

1. Cella di estrazione microbica: <ol><li> Omogeneizzare stuoie microbica con un pestello sterile macinazione mescolando accuratamente. Mettere circa 30 g tutti (peso a umido) di materiale opaco omogeneizzato in un contenitore sterile di Waring frullatore, aggiungere circa 100 ml di 1 M NaCl (o una specifica concentrazione di campione in uso), e si fondono tre volte a velocità media per 1 minuto con raffreddamento intermittente in un congelatore di -20 ° C per 1 min. Trasferire l&#39;impasto in una bottiglia da 250 ml centrifuga e riempire il volume vuoto rimasto con 1 M di NaCl. Preparare la soluzione di NaCl in acqua autoclavata DI e filtro sterilizzarlo. </li><li> Ulteriori rimuovere le cellule microbiche dalla matrice agitando (150 giri) con un vortex (Vortex-Genie R 2) a temperatura ambiente per 30 min. </li><li> Centrifugare a bassa velocità (500x g) per 15 minuti a 4 ° C. Delicatamente trasferire il surnatante in un pallone con il minimo disturbo per il sedimento. </li><li> Utilizzando il sedimento pellet, ripetere la miscelazione e punti 1.2 e 1.3 quattro volte supplementare con l&#39;aggiunta di NaCl fresca ad ogni sedimento pellet alla fine del passo 1.3. Il supernatante di ogni estrazione NaCl viene trasferita in un pallone fresca. </li><li> Combinare i surnatanti dalla cella di 5 estrazioni e centrifugare in aliquote da 200 ml ad alta velocità (25.000 xg) per 15 minuti a 4 ° C. Eliminare il surnatante, risospendere ogni pellet di cellule in 10 ml di esametafosfato di sodio al 2%, si combinano pellet e portare il volume a 200 ml con esametafosfato di sodio al 2%. Lavare le cellule agitando (150 rpm) a temperatura ambiente per 30 min. Al termine di questa fase si dovrebbe avere un solo tubo di cellule per procedere alla fase successiva. </li><li> Centrifuga ad alta velocità (25.000 xg) per 15 minuti a 4 ° C. Eliminare il surnatante, risospendere il pellet in 200 ml di TE (50 mM EDTA), e lavare le cellule agitando (150 rpm) a temperatura ambiente per 10 min. </li><li> Centrifuga ad alta velocità (25.000 xg) per 15 minuti a 4 ° C, scartare il surnatante, risospendere in 15 ml di TE (10 mM EDTA), e memorizzare 200 microlitri aliquote a -80 ° C fino al momento della estrazione del DNA. </li></ol> 2. Estrazione e purificazione del DNA: <ol><li> Aggiungere 200 ul 5 M NaCl, 200 ul SDS 10%, e 100 ul 14,3 M β-mercaptoetanolo al 200 microlitri aliquota di cellule microbiche. Mescolare delicatamente capovolgendo 4-6 volte. </li><li> Soggetto la miscela a 3 turni di gelo-disgelo con immersione in azoto liquido per 2 minuti seguito da scongelamento in un bagno d&#39;acqua a 65 ° C per 5 min. Estendere scongelamento finale per 10 min. </li><li> Aggiungere 200 ul di acetato di potassio 5 M (pH 5,5) e il luogo in ghiaccio per 10 min. Centrifugare a 10000 xg per 10 minuti a 4 ° C e trasferire il surnatante in una nuova provetta utilizzando un ampio tunnel punta della pipetta. Aggiungere 3 ml di RNasi A e incubare a 37 ° C per 1 ora. </li><li> Aggiungere uguale volume di cloroformio, agitare per inversione, e centrifugare a 15000 xg per 10 minuti a temperatura ambiente. </li><li> Con attenzione trasferire lo strato superiore di supernatante in un tubo nuovo usando un ampio calibro punta della pipetta. Evitare di pipettare lo strato bianco di interfaccia si trova tra gli strati superiori e inferiori. Ripetere la procedura di estrazione cloroformio. </li><li> Aggiungere un volume equivalente di acqua refrigerata (a -20 ° C) isopropanolo al surnatante e incubare in ghiaccio per 30 minuti al DNA precipitato. </li><li> Centrifugare alla massima velocità (20.800 xg) per 10 minuti a 4 ° C a pellet di DNA. </li><li> Eliminare il supernatante e lavare il DNA pellet con 1 ml di acqua refrigerata (a -20 ° C) etanolo al 70%. </li><li> Centrifugare alla massima velocità (20.800 xg) per 10 minuti a 4 ° C, scartare il surnatante, ripetere lavare etanolo al 70%, e aria secca DNA pellet per 10 minuti. Ripetere il lavaggio etanolo al 70%. </li><li> Risospendere DNA in 25 microlitri TE (10 mM EDTA), caldo a 65 ° C per 5 minuti, si combinano in un tubo, se i tubi multipli sono stati utilizzati, e make-up del volume di 500 microlitri di TE. </li><li> Aggiungere 500 microlitri ghiacciata (o 4 ° C) polietilene glicole 20% (PEG) (preparato in 1,2 M di NaCl). Mescolare delicatamente per inversione, incubare in ghiaccio per 10 minuti, e centrifugare alla massima velocità per 10 minuti a 4 ° C a pellet di DNA. Rimuovere e scartare il surnatante e lavare il DNA pellet con 1 ml di acqua refrigerata etanolo al 70%, aria secca per 10 minuti, e risospendere nel 30-50 microlitri TE o molecolare dell&#39;acqua grado. Facilitare la risospensione del DNA da riscaldamento a 65 ° C per 5 minuti e il DNA Conservare a -80 ° C. </li></ol> 3. DNA purezza, concentrazione e determinazione Dimensione: <ol><li> Determinare la qualità del DNA misurando la A 260/280 e 260/230 Un rapporto con un NanoDrop 1000 spettrofotometro o qualsiasi altra strumentazione appropriata. </li><li> Determinare la concentrazione di DNA utilizzando Quant-iT kit di analisi dsDNA secondo le istruzioni del produttore. </li><li> Determinare le dimensioni del DNA tramite elettroforesi impulso gel. Preparare 1% gel con TBE 0.5X e utilizzare i seguenti parametri sul Mapper XA CHEF System; Interruttore orario iniziale di 0,35 s, un tempo di passaggio finale di 7,67 s, e 120 ° incluso angolo per un dislivello di 6,0 V / cm a lineare ramping fattore. Macchia gel con 1 x SYBR Green I per 30 min. Carico tra 700-1000 ng di DNA totale su gel. Vedi fig. 3 per una forma condensata del protocollo completo. </li></ol> Rappresentante dei risultati: Celle di estrazione: Sequenziale estratti di cellule microbiche (surnatanti) hanno dimostrato che la torbidità di estratti diminuisce il numero di aumento di estrazioni. Ciò suggerisce una riduzione del numero di cellule a seguito di estrazione ogni cella successiva. Importante da notare qui è che, come ogni ulteriore fase di estrazione delle cellule offre l&#39;opportunità per l&#39;introduzione di contaminanti, il numero di estrazioni cella dovrebbe essere ridotto al minimo in modo che il finale pellet cellulare è rappresentativo della comunità microbica totale. Altre fonti di contaminazione sono stati controllati con l&#39;adozione di adeguate tecniche di laboratorio sterile. Per esempio, le soluzioni sono state preparate in autoclave acqua deionizzata e filtro sterilizzato. I contenitori sono stati sterilizzati con alcol, in autoclave, e trattati alla luce UV ed reticolante ultravioletti. DNA, la concentrazione e la determinazione di qualità: Il protocollo ha dato circa 2 mg di HMW DNA (35-50 kb) (Fig. 4) per grammo di campione tappeto con un 260/280 Un rapporto di 1,6, e una A 260/230 rapporto di 0,7 (Tabella 1). Anche se la A 260/230 rapporto sembra essere bassa, non l&#39;inibizione della valle molecolare-based come la PCR-amplificazione del gene 16S rRNA è stata osservata in uno studio separato 7. E &#39;importante notare che il DNA da hypersaline stuoie ha due principali fonti di contaminazione; EPS e sali. E &#39;quindi possibile che le tracce di questi contaminanti possono influenzare la A 260/230 rapporti nonostante gli enormi sforzi per ridurre i loro effetti sulle applicazioni a valle. DNA stabilire la misura: Elettroforesi su gel campo Pulse utilizza un flusso pulsante con intermittente interruttori direzionale con conseguente striscio DNA, come mostrato in figura 3. Il nostro protocollo ha prodotto un DNA HMW di circa 35-50 kb. Mentre alcune dimensioni del DNA può essere inferiore a 30 kb, è importante avere una parte dello striscio sopra i 35 kb, dato che la clonazione fosmid richiede ~ 40 kb inserti di DNA e frammenti di DNA più grandi offrono un maggiore accesso alle vie biosintetiche intatto. Nei nostri studi, lo striscio DNA superiori a 35 kb è stato asportato e purificato per la grande inserto clonazione vettoriale e altre applicazioni molecolare. <img src="/files/ftp_upload/2887/2887fig1.jpg" alt="Figura 1"/> Figura 1. Ipersalina microbica campionamento sito mat (Big Pond) che si trova su Eleuthera, Bahamas. <img src="/files/ftp_upload/2887/2887fig2.jpg" alt="Figura 2"/> Figura 2. Una sezione del tappeto microbica hypersaline utilizzato in questo studio. Il tappetino microbica è stato ottenuto da uno stagno hypersaline situato su Eleuthera, Bahamas. <img src="/files/ftp_upload/2887/2887fig3.jpg" alt="Figura 3"/> Figura 3. Rappresentazione schematica di procedure necessarie per l&#39;estrazione delle cellule microbiche, la lisi cellulare, estrazione e purificazione del DNA metagenomica. <a href="http://www.jove.com/files/ftp_upload/2887/2887fig3large.jpg">Clicca qui per visualizzare l&#39;immagine ingrandita.</a> <img src="/files/ftp_upload/2887/2887fig4.jpg" alt="Figura 4"/> Figura 4. Caratterizzazione peso molecolare del DNA estratto mediante elettroforesi su gel campo di impulsi. Corsia M è un marker HMW, e corsie 1 e 2 sono repliche di metagenomica DNA estratto dal materassino Eleuthera hypersaline utilizzando il protocollo di cui sopra. <table border="1"><tbody><tr><td> Metodo di estrazione </td><td></td><td> Concentrazione 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> Tabella 1. Misurazione della concentrazione e della qualità del DNA estratto da microbica tappetino hypersaline.

<ol>
	<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>

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Dato che la rimozione totale di cellule da campioni microbiche complesse e molto diverse tappetino non è pratico, la preoccupazione principale è come le cellule estratte rappresentano la complessiva comunità microbica tappeto. In uno studio precedente, PCR-DGGE analisi dei geni rRNA 16S microbica ha mostrato che le cinque fasi di rimozione delle cellule utilizzate in questo protocollo estratti di cellule che sono rappresentativi della comunità microbica complessiva tappeto 7. Il numero effettivo di estraz...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> β-mercaptoetanolo </td><td> Sigma-Aldrich </td><td> M3148 </td><td></td></tr><tr><td> Polietilene glicole 8000 </td><td> Promega </td><td> V3011 </td><td> 20% in 1,2 M di NaCl </td></tr><tr><td> Acetato di potassio </td><td> Fisher Scientific </td><td></td><td> Fisher Scientific </td></tr><tr><td> Quant-iT Assay kit dsDNA </td><td> Invitrogen </td><td> Q33130 </td><td></td></tr><tr><td> RNasi </td><td> Epicentre </td><td> MRNA092 </td><td></td></tr><tr><td> Cloruro di sodio </td><td> Prodotti chimici BDH </td><td> BDH8014 </td><td> Conc appropriato. </td></tr><tr><td> Sodio dodecil solfato </td><td> Fisher Scientific </td><td> 03-500-509 </td><td> 10% in acqua </td></tr><tr><td> sodio esametafosfato </td><td> EMD chimici </td><td> SX0583-3 </td><td> 2% in acqua </td></tr><tr><td> TBE </td><td> Fisher Scientific </td><td> BP1333-1 </td><td></td></tr><tr><td> CHEF Mapper XA sistema </td><td> Bio-Rad Laboratories </td><td> 170-3670 </td><td></td></tr><tr><td> NanoDrop 1000 spettrofotometro </td><td> Thermo Scientific </td><td> ND-1000 </td><td></td></tr><tr><td> Vortex </td><td> Scientific Industries Inc. </td><td></td><td></td></tr><tr><td> Crosslinker ultravioletti </td><td> UVP </td><td></td><td></td></tr><tr><td> Waring Blender </td><td> Waring laboratorio </td><td> LB10S </td><td></td></tr></tbody></table>

extraction of high molecular weight dna from microbial mats

Analisi di successo e preciso e l&#39;interpretazione dei dati metagenomica dipende l&#39;estrazione efficiente di alta qualità, ad elevato peso molecolare (HMW) del DNA della comunità. Tuttavia, i campioni di mat ambientali costituiscono spesso difficoltà ad ottenere grandi concentrazioni di alta qualità, il DNA HMW. Stuoie microbica hypersaline contengono elevate quantità di sostanze polimeriche extracellulari (EPS) 1 e sali che possono inibire le applicazioni a valle di DNA estratto. Metodi diretti e duri sono spesso utilizzati per l&#39;estrazione del DNA da campioni di refrattari. Questi metodi sono generalmente utilizzati perché l&#39;EPS in stuoie, una matrice adesiva, lega il DNA 2,3 durante la lisi diretta. Come risultato di metodi di estrazione più severe, il DNA viene frammentato in piccole dimensioni 4,5,6. Il DNA diventa così inadeguato per la grande inserto clonazione vettore. Per aggirare queste limitazioni, si riporta un metodo migliore per estrarre il DNA HMW di buona qualità e la quantità di stuoie microbica hypersaline. Abbiamo impiegato un metodo indiretto che coinvolge la separazione di cellule microbiche dalla matrice sfondo opaco attraverso la miscelazione e la centrifugazione differenziale. Una combinazione di procedure meccaniche e chimiche è stato utilizzato per estrarre e purificare il DNA da cellule microbiche estratti. Il nostro protocollo produce circa 2 mg di HMW DNA (35-50 kb) per grammo di campione tappeto, con un 260/280 Un rapporto di 1,6. Inoltre, l&#39;amplificazione dei geni rRNA 16S 7 si evince che il protocollo è in grado di minimizzare o eliminare gli effetti inibitori dei contaminanti. I nostri risultati forniscono una metodologia adeguata per l&#39;estrazione del DNA da HMW stuoie microbica per gli studi funzionali metagenomica e può essere applicabile ad altri campioni ambientali dai quali l&#39;estrazione del DNA è una sfida.

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

Forniamo un protocollo migliore per l&#39;estrazione del DNA alto peso molecolare da stuoie microbica hypersaline. Cellule microbiche sono separate dalla matrice tappetino prima dell&#39;estrazione del DNA e la purificazione. Questo aumenta la concentrazione, la qualità e le dimensioni del DNA. Il protocollo può essere utilizzato per altri campioni refrattari.

Estrazione di DNA ad alta peso molecolare da Mats microbica

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

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19.4K Views.  Arnold School of Public Health, University of South Carolina. Forniamo un protocollo migliore per l'estrazione del DNA alto peso molecolare da stuoie microbica hypersaline. Cellule microbiche sono separate dalla matrice tappetino prima dell'estrazione del DNA e la purificazione. Questo aumenta la concentrazione, la qualità e le dimensioni del DNA. Il protocollo può essere utilizzato per altri campioni refrattari.

Video: Extraction of High Molecular Weight DNA from Microbial Mats

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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.

Estrazione del DNA da 0,22 Filtri mM Sterivex e cloruro di Cesio densità centrifugazione gradiente

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.

Estrazione di DNA ad alta peso molecolare genomico da terreni e sedimenti

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.

Estrazione DNA da materiale in paraffina per le analisi genetiche ed epigenetiche

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.

Un ibrido metodo di estrazione del DNA per la valutazione delle comunità batteriche qualitativa e quantitativa di pollame campioni di produzione

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.

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


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

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.

Preparazione core tessuto inclusi in paraffina fissati in formalina per entrambi RNA e DNA Extraction

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.

Metodi modificati per il caricamento di piastre di estrazione del DNA ad alta velocità effettiva riducono il potenziale di contaminazione

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.

Isolamento di miociti atriali e ventricolari murini di alta qualità per misurazioni simultanee di transitori Ca2+ e corrente di calcio di tipo L

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.

Estrazione su piccola scala di Caenorhabditis elegans DNA genomico

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