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

Dit werk werd ondersteund door Strategic Project en Discovery Subsidies voor JDN van het Natural Sciences and Engineering Research Council of Canada (NSERC).

1. Voorbereiding van de reagentia DNA-SIP vereist het gebruik van de reagentia die moeten worden voorbereid op voorhand van de werkelijke procedure. De aanwijzingen voor het bereiden van elk reagens worden in deze sectie en zijn gewijzigd van een vorige SIP-protocol 1. <ol><li> Cesium chloride (CSCL) oplossing voor het bereiden van SIP hellingen - Voorbereiden van een 7.163 M oplossing CsCl door geleidelijk aan het oplossen van 603,0 g CsCl in gedestilleerd en gedeïoniseerd wat...

<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>

Goed ontwerp van stabiele isotoop-probing experimenten is van cruciaal belang voor het verkrijgen van het label DNA boven de achtergrond ongelabelde gemeenschap. Overwegingen met betrekking tot incubatietijden, substraatconcentraties, incubatieomstandigheden (bv. voedingsstoffen, bodemvocht), cross-voeding en replicatie monster zijn elders 10,18 besproken en adviseren wij de lezer deze publicaties bij het ​​ontwerpen van een SIP-incubatie. Gerelateerd aan de huidige protocol, is het de moeite waard commen...

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		<div>
		<table border="1">
		<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-stabiele isotoop indringende (DNA-SIP) is een krachtige techniek voor het identificeren van actieve micro-organismen die bepaalde koolstofsubstraten en voedingsstoffen te assimileren in cellulaire biomassa. Als zodanig is deze teelt-onafhankelijke-techniek is een belangrijke methode voor het toewijzen van metabolische functie om de diverse gemeenschappen bewoont een breed scala van terrestrische en aquatische milieus. Na de incubatie van een milieu-monster met een stabiele isotoop-gelabelde stoffen, wordt gewonnen nucleïnezuur onderworpen aan dichtheidsgradiënt ultracentrifugatie en de daaropvolgende helling fractionering aan nucleïnezuren van verschillende dichtheden scheiden. Zuivering van DNA van cesium chloride ophaalt gelabeld en ongelabelde DNA voor verdere moleculaire karakterisering (bijvoorbeeld vingerafdrukken, microarrays, kloon bibliotheken, metagenomica). Deze video Jove protocol biedt visuele stap-voor-stap uitleg van het protocol voor de dichtheidsgradiënt ultracentrifugatie, gradiënt fractionering en herstel van gelabelde DNA. Het protocol bevat ook proeven van SIP-gegevens en belicht belangrijke tips en waarschuwingen die moeten worden beschouwd als een succesvolle DNA-SIP-analyse te garanderen.

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

DNA Stable-Isotope Probing DNA-SIP

DNA-stabiele isotoop sonderen is een teelt-onafhankelijke methode voor het identificeren en karakteriseren van actieve gemeenschappen van micro-organismen die in staat zijn gebruik te maken van specifieke substraten. Assimilatie van de ondergrond verrijkt met zware isotoop leidt tot opname van gelabelde atomen in de microbiële biomassa. Dichtheidsgradiënt ultracentrifugatie haalt gelabeld DNA voor de downstream-moleculaire analyses.

DNA-Stabiele Isotopen sondering (DNA-SIP)

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

Peilen naar Mitochondriale complexe activiteit in menselijke embryonale stamcellen

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

Methylated DNA Immunoprecipitation

Gemethyleerd DNA Immunoprecipitatie

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

Directe observatie van Enzymen repliceren DNA met behulp van een Single-molecule DNA Stretching Assay

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

Electroeluting DNA Fragments

Electroeluting DNA-fragmenten

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

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

Iteratieve optimalisatie van DNA-duplexen voor kristallisatie van SeqA-DNA complexen

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

Identificatie van metabool actieve bacteriën in de darm van de Generalist<em&gt; Spodoptera littoralis</em&gt; Via DNA Stabiele Isotopen Onderzoek naar gebruik van<sup&gt; 13</sup&gt; C-Glucose

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

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Bereiding van DNA verknoopt polyacrylamide hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

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

Real-time beeldvorming van Single Engineered RNA-transcripten in levende cellen door gebruik Ratiometrische Bimoleculaire Beacons

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

Verlaagde zwaartekracht Milieu Hardware Demonstraties van een Prototype Geminiaturiseerde doorstroomcytometer en Companion Microfluïdische Mixing Technology

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

Probing hoge dichtheid functioneel eiwit Microarrays om eiwit-eiwit interacties Detect

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