Name: bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton
Uploaded: 2011-09-10T14:00:00
Description: Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Il finanziamento di questo progetto è stato fornito dal National Science Foundation concede OCE1029607 (a XM) e MCB0702125 (a MAM) e Gordon and Betty Moore Foundation (a MAM).

1. Acqua esempio per l&#39;elaborazione <ol><li> Filtrare l&#39;acqua 10L ambientale attraverso 1 micron-pori di dimensioni filtri a membrana per rimuovere le particelle più grandi e bacteriovores. Raccogliere il filtrato l&#39;acqua in una damigiana. </li><li> Trasferire 36 ml ciascuno filtrato in 3 provette sterili Eppendorf (50 ml) contenente 4 ml di soluzione appena preparata paraformaldeide (PFA; 10%). Incubare per 2 ore a temperatura ambiente per preservare le cellule. Raccogliere le cellule su 0,22-micron-pori...

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Il nostro approccio coppie BrdU incorporazione, FACS analisi e 16S rDNA per consentire l&#39;identificazione delle specie a livello di batterioplancton che metabolizzano i singoli componenti DOC in ambienti acquatici. Il saggio di incorporazione di BrdU etichette cellule batteriche sulla base di attività metabolica, che permette l&#39;analisi solo sui batteri attivi e quindi non include le cellule dormienti. Nel nostro approccio, incorporazione di BrdU è nei batteri in situ immunodetected e che hanno...

<table border="1"><tbody><tr><td> Nome </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> BrdU </td><td> Reagente </td><td> Sigma </td><td> B5002-5G </td><td></td></tr><tr><td> Lisozima </td><td> Reagente </td><td> Sigma </td><td> L6876-5G </td><td></td></tr><tr><td> Proteinasi K </td><td> Reagente </td><td> Sigma </td><td> P2308-25mg </td><td></td></tr><tr><td> In Situ Kit proliferazione delle cellule, FLUOS </td><td> Kit </td><td> Roche </td><td> 11810740001 </td><td> Consumano più di Anti-BrdU-FLUOS e buffer di incubazione per reazione rispetto a quanto suggerito dal produttore. </td></tr><tr><td> Frame-Seal incubazione Chambers </td><td> Materiale </td><td> Bio-Rad </td><td> SLF-1201 </td><td></td></tr><tr><td> I filtri a membrana in policarbonato (142 mm di diametro, 1,0 micron-poro-size) </td><td> Materiale </td><td> Millipore </td><td> FALP14250 </td><td></td></tr><tr><td> I filtri a membrana in policarbonato (25-mm di diametro, 0,2 micron a poro-size) </td><td> Materiale </td><td> Millipore </td><td> FGLP02500 </td><td></td></tr><tr><td> illustrazioni PuReTaq Ready-To-Go PCR Beads </td><td> Kit </td><td> GE Healthcare </td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquick gel kit di estrazione </td><td> Kit </td><td> QIAGEN </td><td> 28704 </td><td></td></tr><tr><td> FailSafe PCR System </td><td> Kit </td><td> Epicentre </td><td> FS99060 </td><td></td></tr></tbody></table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

I microbi sono agenti principali mediare la degradazione di numerosi carbonio organico disciolto (DOC) substrati in ambienti acquatici. Tuttavia, l&#39;identificazione di taxa batterici che trasformano piscine specifiche di DOC in natura rappresenta una sfida tecnica. Qui si descrive un approccio che le coppie bromodeossiuridina (BrdU) l&#39;incorporazione, fluorescenza cell sorting attivato (FACS) e 16S rRNA gene-based analisi molecolare che consente di cultura indipendente identificazione di batterioplancton in grado di degradare un composto DOC specifico in ambienti acquatici. Microcosmi batterioplancton tre esemplari sono impostati per ricevere sia BrdU e un composto modello DOC (DOC emendamenti), oppure solo BrdU (senza aggiunta di controllo). Sostituti BrdU le posizioni di timidina nel DNA di nuova sintesi batterica e BrdU marcato DNA può essere facilmente immunodetected 1,2. Attraverso una 24-ore di incubazione, batterioplancton che sono in grado di utilizzare il composto aggiunto DOC dovrebbero essere selettivamente attivato, e quindi hanno più alti livelli di incorporazione di BrdU (cellule HI) che non rispondono le cellule negli emendamenti DOC e le cellule nel no- Inoltre controlla (basso cellule incorporazione di BrdU, le cellule LI). Dopo immunolocalizzazione fluorescenza, le cellule si distinguono HI e fisicamente separato dalle cellule LI da fluorescenza separazione delle cellule attivate (FACS) 3. Ordinati DOC-responsive cellule (cellule HI) vengono estratte per il DNA e tassonomicamente individuate attraverso successive rRNA 16S a base genetica tra cui analisi di PCR, la costruzione della libreria clone e sequenziamento.

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Batterioplancton ambientali vengono incubate con un modello di carbonio organico disciolto (DOC) e un reattivo composto di etichettatura del DNA, bromodeossiuridina (BrdU). In seguito, DOC-degradante celle sono separate dalla comunità di massa in base alla loro elevata incorporazione di BrdU usando fluorescenza separazione delle cellule attivate (FACS). Queste cellule vengono poi identificati da successive analisi molecolari.

Bromodeossiuridina (BrdU) Etichettatura e successive ordinamento fluorescenza cellulare attivo per la cultura indipendente Identificazione di carbonio organico disciolto-degradanti batterioplancton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

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Biology

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

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