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

עבודה זו נתמכה על ידי פרויקט אסטרטגי ומענקים דיסקברי JDN מן למדעי הטבע וההנדסה מועצת המחקר של קנדה (NSERC).

1. הכנה של ריאגנטים DNA-SIP מחייב שימוש ריאגנטים כי צריכים להיות מוכנים מראש של ההליך בפועל. הנחיות להכנת כל אחד מגיב המפורטים בסעיף זה שונו מן קודמת SIP פרוטוקול 1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צזיום כלוריד (Cs...

<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>

תכנון נכון של איזוטופ יציב ניסויים חיטוט היא בעלת חשיבות מכרעת לקבלת DNA שכותרתו לעיל הקהילה רקע תווית. שיקולים הקשורים מדגם פעמים הדגירה, ריכוז המצע, תנאי הדגירה (חומרים מזינים כגון לחות תוכן אדמה), בין האכלה שכפול נדונו במקום אחר 10,18 ו - אנו ממליצים להתייעץ עם הק?...

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		<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>
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<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>
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dna stable-isotope probing (dna-sip)

DNA-איזוטופ יציב חיטוט (DNA-SIP) היא טכניקה רבת עוצמה לזיהוי מיקרואורגניזמים פעיל להטמיע מצעים פחמן מסוים וחומרים מזינים לתוך ביומסה הסלולר. ככזה, זו טכניקה טיפוח עצמאית כבר מתודולוגיה חשוב עבור הקצאת פונקציה מטבולית של קהילות מגוונות המאכלסים מגוון רחב של סביבות יבשתי מימיים. לאחר דגירה של המדגם סביבתי עם איזוטופ יציב, תרכובות שכותרתו, חומצות גרעין חילוץ נתונה ultracentrifugation צפיפות שיפוע חלוקה הדרגתי לאחר נפרד חומצות גרעין של צפיפויות שונות. טיהור של ה-DNA של צזיום כלוריד מאחזר שכותרתו תווית ה-DNA לאפיון מולקולרי הבאים (לדוגמה, טביעת אצבע microarrays, ספריות שיבוט, metagenomics). פרוטוקול זה מספק וידאו יופיטר חזותי שלב אחר שלב ההסברים של פרוטוקול עבור שיפוע צפיפות חלוקה ultracentrifugation שיפוע, ושחזור של ה-DNA מסומן. הפרוטוקול כולל גם נתונים מדגם SIP ומדגיש טיפים חשובים מזהיר כי יש לקחת בחשבון כדי להבטיח ניתוח מוצלח SIP-DNA.

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

DNA Stable-Isotope Probing DNA-SIP

DNA-איזוטופ יציב חיטוט היא שיטת טיפוח עצמאית לזהות ולאפיין הקהילות פעיל של מיקרואורגניזמים המסוגלים ניצול מצעים ספציפיים. הטמעת מצע מועשר באיזוטופ הכבד מוביל התאגדות של אטומים שכותרתו לתוך ביומסה של חיידקים. שיפוע ultracentrifugation צפיפות מאחזר DNA שכותרתו עבור ניתוחים מולקולריים במורד הזרם.

DNA-איזוטופ יציב גשוש (ה-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

גשוש עבור פעילות קומפלקס מיטוכונדריאלי של בתאי גזע עובריים אנושיים

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

Methylated DNA Immunoprecipitation

ה-DNA מפוגל 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

תצפית ישירה של ה-DNA אנזימים שכפול באמצעות DNA מולקולה בודדת מתיחה 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 קטעי DNA

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

איטרטיבי אופטימיזציה של דופלקסים DNA להתגבשות של מכלולי SeqA-DNA

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

זיהוי של חיידקים פעילים מטבולית במעיים של Generalist<em&gt; Spodoptera littoralis</em&gt; באמצעות איזוטופ יציב DNA גשוש שימוש<sup&gt; 13</sup&gt; C-גלוקוז

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

הכנת-crosslinked DNA polyacrylamide הידרוג

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

הדמיה בזמן אמת של יחיד תכנון הנדסי RNA תמלילים בתאים חיים באמצעות משואות bimolecular רציומטרי

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

הפגנות חומרת הסביבה מופחתת כובד של טכנולוגיה cytometer זרימה וCompanion Microfluidic אב טיפוס מוקטנת ערבוב

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

חיטוט Microarrays חלבון פונקציונלי בצפיפות גבוהה לזיהוי חלבונים אינטראקציות

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