Ce travail a été financé par la National Science Foundation Programme génomique environnementale (Subvention n ° 0723707 EF-).

1. Extraction de cellule microbienne: <ol><li> Homogénéiser les tapis microbiens avec un pilon stériles meulage en mélangeant soigneusement. Placer environ tous les 30 g (poids humide) de matériau mat homogénéisé dans un contenant stérile d&#39;un mélangeur Waring, ajouter environ 100 ml de NaCl 1 M (ou une concentration spécifique à l&#39;échantillon en utilisation), et mélanger à trois reprises à vitesse moyenne pendant 1 min avec refroidissement intermittent dans un congélateur à -20 ° C pendant 1 min. Transférer la pâte dans une bouteille de 250 mL centrifugeuse et de remplir le volume restant vide avec 1 M de NaCl. Préparer une solution de NaCl dans l&#39;autoclave DI eau et filtrer le stériliser. </li><li> En outre déloger les cellules microbiennes de la matrice par agitation (150 rpm) en utilisant un Vortexer (Vortex-Genie R 2) à température ambiante pendant 30 min. </li><li> Centrifuger à basse vitesse (500x g) pendant 15 min à 4 ° C. Délicatement le transfert du surnageant dans un flacon avec une perturbation minimale de la sédimentation. </li><li> En utilisant les sédiments granulés, répétez les étapes de mélange et 1,2 et 1,3 quatre fois supplémentaires avec l&#39;ajout de frais de NaCl à chaque pastille sédiments à la fin de l&#39;étape 1.3. Le surnageant de chaque extraction de NaCl est transféré dans un flacon de frais. </li><li> Combinez les surnageants des extractions de cellule et centrifuger 5 aliquots de 200 ml à haute vitesse (25 000 xg) pendant 15 min à 4 ° C. Jeter le surnageant, resuspendre chaque culot cellulaire dans 10 ml d&#39;hexamétaphosphate de sodium à 2%, mélanger boulettes et faire monter le volume à 200 ml avec hexamétaphosphate de sodium à 2%. Laver les cellules par la secousse (150 rpm) à température ambiante pendant 30 min. A la fin de cette étape, vous devriez avoir un seul tube de cellules pour procéder à l&#39;étape suivante. </li><li> Centrifugeuse à vitesse élevée (25 000 xg) pendant 15 min à 4 ° C. Jeter le surnageant, resuspendre le culot cellulaire dans 200 ml de TE (50 mM d&#39;EDTA), et laver les cellules par agitation (150 rpm) à température ambiante pendant 10 min. </li><li> Centrifugeuse à vitesse élevée (25 000 xg) pendant 15 min à 4 ° C, éliminer le surnageant, remettre en suspension dans 15 ml de TE (10 mM d&#39;EDTA), et stocker 200 uL d&#39;aliquotes à -80 ° C jusqu&#39;au moment de l&#39;extraction d&#39;ADN. </li></ol> 2. Extraction de l&#39;ADN et purification: <ol><li> Ajouter 200 ul 5 M de NaCl, 200 ul SDS à 10%, et 100 uL 14,3 M β-mercaptoéthanol à l&#39;aliquote de 200 uL microbienne cellule. Mélanger doucement en inversant 4-6 fois. </li><li> Objet le mélange à trois tours de gel-dégel en submergeant dans l&#39;azote liquide pendant 2 min puis décongélation dans un bain d&#39;eau à 65 ° C pendant 5 min. Étendre finale dégel à 10 min. </li><li> Ajouter 200 ul d&#39;acétate de potassium 5 M (pH 5,5) et le placer sur la glace pendant 10 min. Centrifuger à 10000 xg pendant 10 min à 4 ° C et le transfert du surnageant dans un nouveau tube à l&#39;aide d&#39;une pipette de grand diamètre. Ajouter 3 pi de RNase A et incuber à 37 ° C pendant 1h. </li><li> Ajouter un volume égal de chloroforme, agiter brièvement par inversion, et centrifuger à 15 000 xg pendant 10 min à température ambiante. </li><li> Soigneusement transfert de la couche supérieure de surnageant dans un nouveau tube à l&#39;aide d&#39;une pipette de grand diamètre. Évitez de pipetage la couche d&#39;interface blanche située entre les couches supérieure et inférieure. Répétez la procédure chloroforme-extraction. </li><li> Ajouter un volume égal de froid (à -20 ° C) d&#39;isopropanol au surnageant et incuber sur glace pendant 30 min pour précipiter l&#39;ADN. </li><li> Centrifuger à vitesse max (20 800 xg) pendant 10 min à 4 ° C à culot d&#39;ADN. </li><li> Jeter le surnageant et laver l&#39;ADN culot avec 1 mL de froid (à -20 ° C) 70% d&#39;éthanol. </li><li> Centrifuger à vitesse max (20 800 xg) pendant 10 min à 4 ° C, éliminer le surnageant, répétez lavage à l&#39;éthanol 70%, et sécher à l&#39;air culot d&#39;ADN pendant 10 min. Répétez lavage à l&#39;éthanol 70%. </li><li> Resuspendre l&#39;ADN dans 25 ul de TE (10 mM d&#39;EDTA), chaude à 65 ° C pendant 5 min, mélanger dans un tube, si plusieurs tubes ont été utilisés, et le maquillage volume à 500 ul avec TE. </li><li> Ajouter 500 ul glacée (ou à 4 ° C) de polyéthylène glycol de 20% (PEG) (préparé en 1.2 M de NaCl). Mélanger doucement par inversion, incuber sur de la glace pendant 10 min et centrifuger à vitesse maximale pendant 10 min à 4 ° C à culot d&#39;ADN. Retirez et jetez le surnageant et laver l&#39;ADN granulés avec 1 ml d&#39;éthanol à 70% réfrigérés, séchés à l&#39;air pendant 10 min, et remettre en 30-50 uL TE ou moléculaire de l&#39;eau de qualité. Faciliter la remise en suspension de l&#39;ADN par le réchauffement à 65 ° C pendant 5 min et entreposer l&#39;ADN à -80 ° C. </li></ol> 3. Pureté d&#39;ADN, de concentration et de détermination Taille: <ol><li> Déterminer la qualité de l&#39;ADN par la mesure de la 260/280 A et A 260/230 ratios en utilisant un spectrophotomètre ou Nanodrop 1000 toute autre instrumentation appropriée. </li><li> Déterminer la concentration d&#39;ADN en utilisant Quant-iT Kit de dosage ADNdb conformément aux instructions du fabricant. </li><li> Déterminer la taille d&#39;ADN par électrophorèse sur gel d&#39;impulsion sur le terrain. Préparer 1% gel d&#39;agarose avec 0.5X TBE et utiliser les paramètres suivants sur le système CHEF Mapper XA; changer l&#39;heure initiale de 0,35 s, un temps final de passer 7,67 s et 120 ° angle inclus pour une pente de 6,0 V / cm à une r linéairesamplification facteur. Tache de gel avec 1 x SYBR j&#39;ai vert pendant 30 min. Charge entre 700-1000 ng d&#39;ADN total sur gel. Voir Fig. 3 pour une forme condensée du protocole complet. </li></ol> Les résultats représentatifs: Extraction de cellule: Séquentielle des extraits de cellules microbiennes (surnageants) ont montré que la turbidité des extraits diminue à mesure que le nombre d&#39;augmentation des extractions. Ceci suggère une réduction du nombre de cellules après chaque extraction de cellule successives. Important à noter ici est que chaque étape d&#39;extraction de cellules supplémentaires prévoit la possibilité pour l&#39;introduction de contaminants, le nombre d&#39;extractions de cellule doit être minimisée pour que le culot cellulaire final est représentatif de la communauté microbienne globale. D&#39;autres sources de contamination ont été contrôlés par l&#39;adoption de techniques de laboratoire adéquats stérile. Par exemple, les solutions ont été préparées dans de l&#39;eau déminéralisée à l&#39;autoclave et stérilisés par filtration. Conteneurs ont été stérilisés avec de l&#39;alcool, autoclave, et traités sous la lumière UV et réticulant ultraviolets. Concentration de l&#39;ADN et la détermination de la qualité: Le protocole a donné environ 2 pg d&#39;ADN HMW (35-50 ko) (Fig. 4) par gramme d&#39;échantillon tapis avec un A 260/280 ratio de 1,6, et un A 260/230 ratio de 0,7 (tableau 1). Bien que le rapport A 260/230 semblait être faible, pas d&#39;inhibition de l&#39;aval moléculaires à base d&#39;application tels que la PCR d&#39;amplification des gènes d&#39;ARNr 16S a été observée dans une étude distincte 7. Il est important de noter que l&#39;ADN de hypersalin tapis a deux principales sources de contamination; EPS et des sels. Il est donc possible que des traces de ces contaminants peuvent influencer l&#39;260/230 Un ratio en dépit des efforts énormes pour réduire leurs effets sur les applications en aval. L&#39;ADN détermination de la taille: Électrophorèse en champ pulsé utilise un débit pulsé courant avec interrupteurs intermittents directionnelle traduit par un frottis d&#39;ADN comme le montre la figure 3. Notre protocole donné un ADN HMW d&#39;environ 35 à 50 ko. Alors que certains de taille d&#39;ADN peut être inférieur à 30 ko, il est important d&#39;avoir une partie du frottis-dessus de 35 ko, étant donné que le clonage fosmide nécessite environ 40 kb inserts d&#39;ADN et des fragments d&#39;ADN plus de fournir un meilleur accès aux voies de biosynthèse intacte. Dans nos études, le frottis d&#39;ADN de plus de 35 kb a été excisée et purifiée pour le vecteur de clonage à grande insertion et d&#39;autres applications moléculaires. <img src="/files/ftp_upload/2887/2887fig1.jpg" alt="Figure 1"/> Figure 1. Microbienne hypersalin tapis de site d&#39;échantillonnage (Big Pond) situé sur Eleuthera, aux Bahamas. <img src="/files/ftp_upload/2887/2887fig2.jpg" alt="Figure 2"/> Figure 2. Une coupe transversale du tapis microbien hypersalin utilisées dans cette étude. Les tapis microbiens a été obtenue à partir d&#39;un étang situé sur hypersalin Eleuthera, aux Bahamas. <img src="/files/ftp_upload/2887/2887fig3.jpg" alt="Figure 3"/> Figure 3. Représentation schématique des procédures impliquées dans l&#39;extraction de cellules microbiennes, la lyse cellulaire, l&#39;extraction et la purification d&#39;ADN métagénomique. <a href="http://www.jove.com/files/ftp_upload/2887/2887fig3large.jpg">Cliquez ici pour voir une image plus grande.</a> <img src="/files/ftp_upload/2887/2887fig4.jpg" alt="Figure 4"/> Figure 4. Caractérisation du poids moléculaire de l&#39;ADN extrait par électrophorèse en champ pulsé. Lane M est un marqueur HMW et des voies 1 et 2 sont de l&#39;ADN se réplique métagénomique extraites du tapis de Eleuthera hypersalin utilisant le protocole ci-dessus. <table border="1"><tbody><tr><td> Méthode d&#39;extraction </td><td></td><td> Concentration 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> Tableau 1. Mesure de la concentration et la qualité de l&#39;ADN extrait de tapis microbiens hypersalin.

<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>

Étant donné que l&#39;élimination totale des cellules du complexe et très diversifié échantillons des tapis microbiens n&#39;est pas pratique, la principale préoccupation est de savoir comment bien les cellules extraites de représenter la communauté microbienne globale tapis. Dans une étude précédente, la PCR-DGGE analyse des gènes microbiens ARNr 16S ont montré que les cinq étapes d&#39;élimination des cellules utilisées dans le présent protocole d&#39;extraits des cellules qui sont représentatifs de...

<table border="1"><tbody><tr><th> Nom de réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> β-mercaptoéthanol </td><td> Sigma-Aldrich </td><td> M3148 </td><td></td></tr><tr><td> Le polyéthylène glycol 8000 </td><td> Promega </td><td> V3011 </td><td> 20% dans 1,2 M de NaCl </td></tr><tr><td> L&#39;acétate de potassium </td><td> Fisher Scientific </td><td></td><td> Fisher Scientific </td></tr><tr><td> Quant-iT Assay Kit ADNdb </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> Chlorure de sodium </td><td> BDH Chemicals </td><td> BDH8014 </td><td> Conc appropriées. </td></tr><tr><td> Dodécyl sulfate de sodium </td><td> Fisher Scientific </td><td> 03-500-509 </td><td> 10% dans de l&#39;eau </td></tr><tr><td> hexamétaphosphate de sodium </td><td> EMD Chemicals </td><td> SX0583-3 </td><td> 2% dans de l&#39;eau </td></tr><tr><td> TBE </td><td> Fisher Scientific </td><td> BP1333-1 </td><td></td></tr><tr><td> CHEF Mapper XA système </td><td> Bio-Rad Laboratories </td><td> 170-3670 </td><td></td></tr><tr><td> NanoDrop 1000 spectrophotomètre </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> Crosslinker ultraviolet </td><td> UVP </td><td></td><td></td></tr><tr><td> Mélangeur Waring </td><td> Waring de laboratoire </td><td> LB10S </td><td></td></tr></tbody></table>

extraction of high molecular weight dna from microbial mats

Analyse réussie et exacte et l&#39;interprétation des données métagénomiques dépend de l&#39;extraction efficace de haute qualité, l&#39;ADN de haut poids moléculaire de la communauté (HMW). Cependant, les échantillons environnementaux posent souvent des tapis de difficultés à obtenir de grandes concentrations de haute qualité, l&#39;ADN HMW. Tapis microbiens hypersalin contiennent des quantités élevées de substances polymériques extracellulaires (EPS) 1 et les sels qui peuvent inhiber les applications en aval de l&#39;ADN extrait. Les méthodes directes et dures sont souvent utilisés dans l&#39;extraction d&#39;ADN à partir d&#39;échantillons réfractaires. Ces méthodes sont généralement utilisées parce que le BPA dans des nattes, une matrice adhésive, lie l&#39;ADN 2,3 lors de la lyse directe. En raison de méthodes d&#39;extraction plus sévères, l&#39;ADN se fragmente en petites tailles 4,5,6. L&#39;ADN devient donc inapproprié pour le vecteur de clonage à grande insérer. Afin de contourner ces limitations, nous présentons une méthodologie améliorée pour en extraire l&#39;ADN HMW de bonne qualité et la quantité de tapis microbiens hypersalin. Nous avons utilisé une méthode indirecte impliquant la séparation des cellules microbiennes de la matrice mat fond par mélange et centrifugation différentielle. Une combinaison de procédés mécaniques et chimiques a été utilisé pour extraire et purifier l&#39;ADN des cellules microbiennes extrait. Notre protocole de rendements d&#39;environ 2 pg d&#39;ADN HMW (35-50 ko) par gramme d&#39;échantillon mat, avec un A 260/280 ratio de 1,6. Par ailleurs, l&#39;amplification des gènes d&#39;ARNr 16S 7 suggère que le protocole est en mesure de minimiser ou éliminer les effets inhibiteurs de contaminants. Nos résultats fournissent une méthodologie appropriée pour l&#39;extraction d&#39;ADN à partir HMW tapis microbiens fonctionnels pour les études métagénomiques et peut être applicable à d&#39;autres échantillons environnementaux à partir de laquelle l&#39;extraction d&#39;ADN est un défi.

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

Nous fournissons un protocole amélioré pour l&#39;extraction ADN de haut poids moléculaire à partir de tapis microbiens hypersalin. Les cellules microbiennes sont séparés de la matrice mat avant l&#39;extraction de l&#39;ADN et de purification. Cela améliore la concentration, la qualité et la taille de l&#39;ADN. Le protocole peut être utilisé pour d&#39;autres échantillons réfractaires.

Extraction d&#39;ADN de haut poids moléculaire de tapis microbiens

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 vues.  Arnold School of Public Health, University of South Carolina. Nous fournissons un protocole amélioré pour l'extraction ADN de haut poids moléculaire à partir de tapis microbiens hypersalin. Les cellules microbiennes sont séparés de la matrice mat avant l'extraction de l'ADN et de purification. Cela améliore la concentration, la qualité et la taille de l'ADN. Le protocole peut être utilisé pour d'autres échantillons réfractaires.

Vidéo: Extraction of High Molecular Weight DNA from Microbial Mats

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Biologie des communautés microbiennes - Interview

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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.

Extraction de l&#39;ADN à partir de 0,22 Filtres Sterivex uM et le césium Centrifugation gradient de chlorure de densité

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.

Extraction d&#39;ADN de haut poids moléculaire génomique par les sols et sédiments

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.

Extraction d&#39;ADN de matériel inclus en paraffine pour les analyses génétiques et épigénétiques

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.

Une méthode hybride d&#39;extraction d&#39;ADN pour l&#39;évaluation qualitative et quantitative des communautés bactériennes de la volaille Échantillons de production

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.

Mucine Agarose Gel Electrophoresis: Western Blot pour les glycoprotéines de haut poids moléculaire,


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.

Préparation de Cores de tissus enrobés de paraffine fixés au formol pour les ARN et d&#39;extraction d&#39;ADN

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.

Méthodes modifiées pour le chargement des plaques d’extraction d’ADN à haut débit réduisent le risque de contamination

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.

Isolement de myocytes auriculaires et ventriculaires murins de haute qualité pour des mesures simultanées des transitoires Ca2+ et du courant calcique de type 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.

Extraction à petite échelle de l’ADN génomique de Caenorhabditis elegans

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