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

Эта работа была поддержана стратегических проектов и Discovery Гранты JDN от естествознания и техники Научно-исследовательский совет Канады (NSERC).

1. Подготовка реагентов ДНК-SIP требует использования реагентов, которые должны быть подготовлены до начала фактической процедуры. Направления подготовки каждого реагента, перечислены в этом разделе и изменяются от предыдущего протокола SIP 1. <ol><li> Хлорид цезия (CsCl)...

<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>

Правильное проектирование стабильных изотопов зондирования экспериментов имеет решающее значение для получения меченых ДНК выше фона немеченого сообщества. Соображения, связанные с образцом времени инкубации, субстрат концентрации, условия инкубации (например, питательных веществ...

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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)

ДНК-стабильный изотоп зондирования (ДНК-SIP) является мощным средством для идентификации активных микроорганизмов, усваивающих частности подложках углерода и питательных веществ в клеточную биомассу. Таким образом, это выращивание независимый метод был важным методологии присвоения метаболические функции различных общин, населяющих широкий диапазон наземных и водных средах. После инкубации экологические образца со стабильным изотопом меченых соединений, выделенных нуклеиновой кислоты подвергается плотность ультрацентрифугирования градиента и последующего фракционирования градиент на отдельные нуклеиновых кислот разной плотности. Очистка ДНК из хлорида цезия извлекает меченого и немеченого ДНК для последующего молекулярная характеристика (например, отпечатков пальцев, микрочипы, клон библиотеки, metagenomics). Это Юпитер протокола видео обеспечивает визуальную шаг за шагом объяснения протокол для градиента плотности ультрацентрифугирования, фракционирования градиента и восстановления меченых ДНК. Протокол также включает в себя образцы SIP данных и выделяет важные советы и предостережения, которые необходимо учитывать для обеспечения успешной ДНК-SIP анализа.

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

DNA Stable-Isotope Probing DNA-SIP

ДНК-стабильный изотоп зондирования выращивание-независимый метод для определения и характеристики активных сообществ микроорганизмов, способных использовать специфические субстраты. Ассимиляция субстрата обогащенный тяжелым изотопом приводит к включению меченых атомов в микробной биомассы. Ультрацентрифугирования градиента плотности извлекает меченых ДНК для последующих молекулярного анализа.

ДНК-Stable-Изотопный Зондирование (ДНК-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

Зондирование для митохондриального комплекса активность в эмбриональных стволовых клеток человека

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

Methylated DNA Immunoprecipitation

Метилированный Иммунопреципитация ДНК

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

Прямое наблюдение Ферменты Репликация ДНК Использование одиночных молекул ДНК Растяжка Пробирной

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 фрагментов ДНК

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

Итерационные Оптимизация дуплексов ДНК для кристаллизации SeqA ДНК-комплексов

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

Идентификация метаболически активных бактерий в кишечнике в Универсал<em&gt; Spodoptera littoralis</em&gt; Через ДНК стабильный изотоп Зондирование Использование<sup&gt; 13</sup&gt; С-Глюкоза

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

Подготовка ДНК-сшитый полиакриламидных гидрогелей

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

В режиме реального времени изображений Единого Engineered РНК-транскриптов в живых клетках, используя Логометрический Бимолекулярные маяки

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

Сниженной гравитационные окружающей среды и комплектующие Демонстрации проточной цитометрии и Companion микрожидкостных Технология смешивания Прототип миниатюрных

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

Исследование высокой плотности белковых Функциональные Microarrays для обнаружения белок-белковых взаимодействий

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