Name: dna stable-isotope probing (dna-sip)
Uploaded: 2010-08-02T17:25:00
Description: Watch this Scientific Journal Video about DNA Stable-Isotope Probing DNA-SIP at JoVE.com

Bu çalışma Stratejik Proje ve Kanada Doğal Bilimler ve Mühendislik Araştırma Konseyi (NSERC) JDN Discovery Hibeler tarafından desteklenmiştir.

1. Reaktiflerin hazırlanması DNA SIP gerçek prosedür önceden hazırlanmalıdır reaktiflerin kullanımını gerektirir. Bu bölümde listelenen her reaktif hazırlanması için yönde ve bir önceki SIP protokolü 1 değiştirilmiş. <ol><li> SIP gradyanlar hazırlanması için Sezyum klorür (CSCL) çözümü yavaş yavaş son hacim 500 mL distile ve deiyonize su CSCL 603,0 g (GKD 2 O) eriterek 7,163 M CSCL çözüm hazırlayın. 500 ml aşmayacak şekilde dikka...

<ol>
	<li>Neufeld, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+stable-isotope+probing.">DNA stable-isotope probing.</a> Nat. Protocols. 2, 860-866 (2007).</li><li>Neufeld, J. D., Boden, R., Moussard, H., Sch&#228;fer, H., Murrell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-specific+clades+of+active+marine+methylotrophs+associated+with+a+phytoplankton+bloom+in+a+temperate+coastal+environment.">Substrate-specific clades of active marine methylotrophs associated with a phytoplankton bloom in a temperate coastal environment.</a> Appl. Environ. Microbiol. 74, 7321-7328 (2009).</li><li>Nercessian, O., Noyes, E., Kalyuzhnaya, M. G., Lidstrom, M. E., Chistoserdova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+populations+active+in+metabolism+of+C1+compounds+in+the+sediment+of+Lake+Washington,+a+freshwater+lake.">Bacterial populations active in metabolism of C1 compounds in the sediment of Lake Washington, a freshwater lake.</a> Appl. Environ. Microbiol. 71, 6885-6899 (2005).</li><li>Padmanabhan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiration+of+13C-labelled+substrates+added+to+soil+in+the+field+and+subsequent+16S+rRNA+gene+analysis+of+13C-labelled+soil+DNA.">Respiration of 13C-labelled substrates added to soil in the field and subsequent 16S rRNA gene analysis of 13C-labelled soil DNA.</a> Appl. Environ. Microbiol. 69, 1614-1622 (2003).</li><li>Bernard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+identification+of+soil+microbial+populations+actively+assimilating+carbon+from+13C-labelled+wheat+residue+as+estimated+by+DNA-+and+RNA-SIP+techniques.">Dynamics and identification of soil microbial populations actively assimilating carbon from 13C-labelled wheat residue as estimated by DNA- and RNA-SIP techniques.</a> Environ. Microbiol. 9, 752-764 (2007).</li><li>Haichar, e. l. Z. a. h. a. r., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cellulolytic+bacteria+in+soil+by+stable+isotope+probing.">Identification of cellulolytic bacteria in soil by stable isotope probing.</a> Environ. Microbiol. 9, 625-634 (2007).</li><li>Addison, S., McDonald, I., Lloyd-Jones, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+probing:+Technical+considerations+when+resolving+15N-labelled+RNA+in+gradients.">Stable isotope probing: Technical considerations when resolving 15N-labelled RNA in gradients.</a> J. Microbiol. Meth. 80, 70-75 (2009).</li><li>Buckley, D. H., Huangyutitham, V., Hsu, S. -. F., Nelson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+probing+with+15N+achieved+by+disentangling+the+effects+of+genome+G+++C+content+and+isotope+enrichment+on+DNA+density.">Stable isotope probing with 15N achieved by disentangling the effects of genome G + C content and isotope enrichment on DNA density.</a> Appl. Environ. Microbiol. 73, 3189-3195 (2007).</li><li>Schwartz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+growing+microorganisms+in+soil+by+stable+isotope+probing+with+H218O.">Characterization of growing microorganisms in soil by stable isotope probing with H218O.</a> Appl. Environ. Microbiol. 73, 2541-2546 (2007).</li><li>Neufeld, J. D., Dumont, M. G., Vohra, J., Murrell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+for+the+use+of+stable+isotope+probing+in+microbial+ecology.">Methodological considerations for the use of stable isotope probing in microbial ecology.</a> Microb. Ecol. 53, 435-442 (2007).</li><li>Martineau, C., Whyte, L., Greer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+SYBR+safe+technique+for+the+sensitive+detection+of+DNA+in+cesium+chloride+density+gradients+for+stable+isotope+probing+assays.">Development of a SYBR safe technique for the sensitive detection of DNA in cesium chloride density gradients for stable isotope probing assays.</a> J. Microbiol. Meth. 73, 199-202 (2008).</li><li>Bartram, A. K., Poon, C., Neufeld, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+contamination+of+glycogen+used+in+nucleic+acid+precipitation+and+assessment+of+linear+polyacrylamide+as+an+alternative+co-precipitant.">Nucleic acid contamination of glycogen used in nucleic acid precipitation and assessment of linear polyacrylamide as an alternative co-precipitant.</a> Biotechniques. 47, 1019-1022 (2009).</li><li>Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+uncultivated+majority:+combining+DNA+stable-isotope+probing,+multiple+displacement+amplification+and+metagenomic+analyses+of+uncultivated+Methylocystis+in+acidic+peatlands.">Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands.</a> Environ. Microbiol. 10, 2609-2622 (2008).</li><li>Neufeld, J. D., Chen, Y., Dumont, M. G., Murrell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+methylotrophs+revealed+by+stable-isotope+probing,+multiple+displacement+amplification+and+metagenomics.">Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics.</a> Environ. Microbiol. 10, 1526-1535 (2008).</li><li>Kalyuzhnaya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+metagenomics+targets+specific+functional+types+in+complex+microbial+communities.">High-resolution metagenomics targets specific functional types in complex microbial communities.</a> Nat. Biotechnol. 26, 1029-1034 (2008).</li><li>Binga, E. K., Lasken, R. S., Neufeld, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Something+from+(almost)+nothing:+the+impact+of+multiple+displacement+amplification+on+microbial+ecology.">Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology.</a> ISME J. 2, 233-241 (2008).</li><li>Green, S. J., Leigh, M. B., Neufeld, J. D., Timmis, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microbiology of Hydrocarbon and Lipid Microbiology. , 4137-4158 (2010).</li><li>Neufeld, J. D., Wagner, M., Murrell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Who+eats+what,+where+and+when?+Isotope-labelling+experiments+are+coming+of+age.">Who eats what, where and when? Isotope-labelling experiments are coming of age.</a> ISME J. 1, 103-110 (2007).</li><li>Gallagher, E., McGuinness, L., Phelps, C., Young, L. Y., Kerkhof, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+shortens+the+incubation+time+needed+to+detect+benzoate-utilizing+denitrifying+bacteria+by+stable-isotope+probing.">DNA shortens the incubation time needed to detect benzoate-utilizing denitrifying bacteria by stable-isotope probing.</a> Appl. Environ. Microbiol. 71, 5192-5196 .</li></ol>

Kararlı izotop sondalama deneyleri doğru tasarım arka plan etiketlenmemiş topluluk yukarıdaki işaretli DNA elde etmek için kritik önem taşımaktadır. Inkübasyon sürelerinde, substrat konsantrasyonları, inkübasyon koşulları (örneğin, besin, toprak nem içeriği), çapraz besleme ve çoğaltma örnek ilgili Hususlar başka 10,18 tartışılan ve bir SIP inkübasyon tasarlarken okuyucu bu yayınların başvurun tavsiye edilmiştir. Geçerli protokol, SIP gradyanlar verilerin yorumlanması ile i...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bromophenol Blue</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>BP115-25</td>
<td>&nbsp;</td>
<tr>
<td>Cesium chloride</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>BP210-500</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol, reagent grade</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>652261</td>
<td>&nbsp;</td>
<tr>
<td>Ethidium bromide</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>E1510</td>
<td>&nbsp;</td>
<tr>
<td>Hydrochloric acid</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>351285212</td>
<td>&nbsp;</td>
<tr>
<td>Linear polyacrylamide</td>
<td>Reagent</td>
<td>Applichem</td>
<td>A6587</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene Glycol 6000</td>
<td>Reagent</td>
<td>VWR</td>
<td>CAPX1286L-4</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Chloride</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>AC42409-0010</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>S2711</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Hydroxide pellets</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>S3181</td>
<td>&nbsp;</td>
<tr>
<td>Tris base</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>BP1521</td>
<td>&nbsp;</td>
<tr>
<td>Dark Reader</td>
<td>Equipment</td>
<td>Clare Chemical</td>
<td>DR46B</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge</td>
<td>Equipment</td>
<td>Eppendorf</td>
<td>5424 000.410</td>
<td>&nbsp;</td>
<tr>
<td>Nanodrop 2000</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>361013650</td>
<td>&nbsp;</td>
<tr>
<td>Infusion pump</td>
<td>Equipment</td>
<td>Braintree Scientific</td>
<td>N/A</td>
<td>Model Number: BSP 
See <a href="http://www.braintreesci.com">www.braintreesci.com</a> for ordering details.</td>
</tr>
<tr>
<td>Tube sealer</td>
<td>Equipment</td>
<td>Beckman-Coulter</td>
<td>358312</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge</td>
<td>Equipment</td>
<td>Beckman-Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ultracentrifuge rotor</td>
<td>Equipment</td>
<td>Beckman-Coulter</td>
<td>362754</td>
<td>&nbsp;</td>
<tr>
<td>Ultraviolet light source</td>
<td>Equipment</td>
<td>UVP Inc. </td>
<td>95-0017-09</td>
<td>Any UV source will suffice</td>
</tr>
<tr>
<td>Ultraviolet light face shield</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>114051C</td>
<td>&nbsp;</td>
<tr>
<td>Butyl rubber stoppers, gray</td>
<td>Material</td>
<td>Sigma-Aldrich</td>
<td>27232</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge tubes</td>
<td>Material</td>
<td>Beckman-Coulter</td>
<td>342412</td>
<td>&nbsp;</td>
<tr>
<td>Hypodermic needle, 23 gauge, 2&#x201D; length</td>
<td>Material</td>
<td>BD</td>
<td>305145</td>
<td>&nbsp;</td>
<tr>
<td>Microfuge tubes, 1.5 mL</td>
<td>Material</td>
<td>DiaMed</td>
<td>AD151-N500</td>
<td>&nbsp;</td>
<tr>
<td>Open center seals, 20 mm diameter</td>
<td>Material</td>
<td>Sigma-Aldrich</td>
<td>27230-U</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipettes, glass</td>
<td>Material</td>
<td>Fisher Scientific</td>
<td>13-678-6C</td>
<td>&nbsp;</td>
<tr>
<td>Pipet tips</td>
<td>Material</td>
<td>DiaMed</td>
<td>BPS340-1000 </td>
<td>Catalogue number is for 200 &mu;l tips. 10 or 20 &mu;l tips may be purchased from the same source</td>
</tr>
<tr>
<td>Pump tubing 1.5 mm bore x 1.5 mm wall</td>
<td>Material</td>
<td>Appleton Woods</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Screw-cap tubes, 15 mL</td>
<td>Material</td>
<td>DiaMed</td>
<td>AD15MLP-S </td>
<td>&nbsp;</td>
<tr>
<td>Serum vials, 125 mL volume</td>
<td>Material</td>
<td>Sigma-Aldrich</td>
<td>Z114014</td>
<td>&nbsp;</td>
<tr>
<td>Syringe, 60 mL</td>
<td>Material</td>
<td>BD</td>
<td>309653</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

dna stable-isotope probing (dna-sip)

DNA stabil izotop (DNA-SIP) problama özellikle hücresel biyokütle içinde karbon yüzeyleri ve besin asimile aktif mikroorganizmaların belirlenmesi için güçlü bir tekniktir. Bunun gibi, bu, yetiştirme bağımsız tekniği, geniş bir yelpazede karasal ve sucul ortamlarda yaşayan farklı toplulukların metabolik fonksiyonu atama için önemli bir yöntem olmuştur. Istikrarlı izotop etiketli bileşikler ile çevresel bir örnek inkübasyon ardından, çıkarılan nükleik asit, nükleik asitlerin farklı yoğunlukları ayrı bir yoğunluk gradiyenti Ultrasantrifügasyon ve sonraki degrade fraksiyonasyon tabi tutulur. Sezyum klorür DNA saflaştırılması sonraki moleküler karakterizasyonu (örneğin parmak izi, mikroarrayler, klon kütüphaneler, metagenomics) DNA etiketli ve etiketlenmemiş alır. Bu vallahi video protokol yoğunluk gradiyenti Ultrasantrifügasyon, degrade fraksiyonasyon ve işaretli DNA kurtarma için protokol görsel olarak adım adım açıklamalar sağlar. Protokol, aynı zamanda örnek SIP veri ve golleri önemli ipuçları içerir ve başarılı bir SIP DNA analizi sağlamak için düşünülmesi gerektiğini uyarıyor.

Watch this Scientific Journal Video about DNA Stable-Isotope Probing DNA-SIP at JoVE.com

DNA Stable-Isotope Probing DNA-SIP

DNA kararlı izotop problama özel substratlar kullanma yeteneğine sahip olan mikroorganizmaların aktif toplulukları tanımlamak ve karakterize etmek için bir kültür bağımsız bir yöntemdir. Ağır izotop zenginleştirilmiş substrat Asimilasyon etiketli atomların mikrobiyal biyokütle içine dahil yol açar. Yoğunluk degrade Ultrasantrifügasyon downstream moleküler analizler için işaretli DNA alır.

DNA Kararlı-İzotop (DNA-SIP) Probing

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

Research

JoVE Journal

Biology

DNA Stable-Isotope Probing (DNA-SIP)

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

İnsan Embriyonik Kök Hücreleri Mitokondrial Kompleksi Aktivite araştırmayı

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Biyoloji

Methylated DNA Immunoprecipitation

Metillenmiş DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

Enzimlerin Klonlanması DNA Doğrudan Gözlem Tek bir molekülü DNA Testi Esneme

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Electroeluting DNA Fragments

DNA Fragments Electroeluting

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

RNA içeren Oligonükleotidler kullanarak Dönüşüm Genomik DNA, RNA / DNA Hibritlerin Üretimi

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

SeqA-DNA kompleksleri kristalleşmesi için DNA dupleksler optimizasyonu İteratif

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Kültürlü ve Gut metabolik Aktif Bakterilerin Tanımlanması<em&gt; Spodoptera littoralis</em&gt; DNA Kararlı İzotop yoluyla kullanma Probing<sup&gt; 13</sup&gt; C = Glukoz

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Prototip Minyatür Akış Sitometresi ve Companion mikroakışkan Karıştırma Teknoloji İndirimli-gravite Çevre Donanım Gösterileri

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Yüksek yoğunluklu Fonksiyonel Protein mikrodizileri Probing Protein-protein Etkileşimleri Algılama

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...