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

Bu projenin finansmanı Ulusal Bilim Vakfı hibe OCE1029607 (XM) ve MCB0702125 (MAM) ve Gordon ve Betty Moore Vakfı (MAM) tarafından sağlandı.

1. Su örneği işleme <ol><li> Filtre 1 mikron gözenek boyutlu membran filtreler aracılığıyla 10L çevre su büyük parçacıklar ve bacteriovores kaldırın. Bir damacana su süzüntü toplayın. </li><li> Transferi 3 4 ml taze hazırlanmış paraformaldehid çözüm (% 10 PFA) içeren steril Eppendorf tüpleri (50 ml) içine 36 ml süzüntü her. Hücreleri korumak için 2 saat oda sıcaklığında inkübe edin. Vakum filtrasyon ile 0.22 mikron-gözenek boyutlu beyaz membran filtreler üzerine hücreleri topla...

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Bizim yaklaşımımız çiftler BrdU bir FACS ve 16S rDNA analiz sucul ortamlarda bireysel DOC bileşenleri metabolize bacterioplankton tür düzeyinde kimlik izin vermek. BrdU dahil tahlil bakteri hücrelerinin metabolik faaliyetleri, sadece aktif bakteri analizi sağlar ve böylece uyuyan hücreleri içermez dayalı etiketler. Bizim yaklaşımımız, BrdU dahil, sonradan görselleştirildiği BrdU esas farklı düzeylerde immunodetected yerinde ve bakteri FACS kullanarak gruplandırılmış ve sıralanmış. ...

<table border="1"><tbody><tr><td> Isim </td><td> Tip </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> BrdU </td><td> Reaktif </td><td> Sigma </td><td> B5002-5G </td><td></td></tr><tr><td> Lizozim </td><td> Reaktif </td><td> Sigma </td><td> L6876-5G </td><td></td></tr><tr><td> Proteinaz K </td><td> Reaktif </td><td> Sigma </td><td> P2308-25mg </td><td></td></tr><tr><td> Situ Hücre Çoğalması Takımı, FLUOS </td><td> Seti </td><td> Roche </td><td> 11810740001 </td><td> Anti-BrdU-FLUOS ve üretici tarafından önerilen daha reaksiyon başına Kuluçka tampon daha fazla tüketin. </td></tr><tr><td> Çerçeve-Seal Kuluçka Odaları </td><td> Malzeme </td><td> Bio-Rad </td><td> SLF-1201 </td><td></td></tr><tr><td> Polikarbonat Membran Filtreler (142 mm çap, 1,0 mikron gözenek boyutu) </td><td> Malzeme </td><td> Millipore </td><td> FALP14250 </td><td></td></tr><tr><td> Polikarbonat Membran Filtreler (25 mm çap, 0,2 mikron gözenek boyutu) </td><td> Malzeme </td><td> Millipore </td><td> FGLP02500 </td><td></td></tr><tr><td> versiyonu gösterime PuReTaq Ready-To-Go PCR boncuklar </td><td> Seti </td><td> GE Healthcare </td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquick jel ekstraksiyon kiti </td><td> Seti </td><td> QIAGEN </td><td> 28704 </td><td></td></tr><tr><td> FailSafe PCR Sistemi </td><td> Seti </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

Mikroplar sucul ortamlarda pek çok çözünmüş organik karbon (DOC) yüzeylerde bozulması aracılık önemli ajanlardır. Ancak, doğada DOC özel havuzları dönüştürmek bakteri taksonların belirlenmesi teknik bir sorun teşkil etmektedir. Burada bir yaklaşım tarif çiftler bromodeoxyuridine (BrdU) dahil, floresan aktif hücre sıralama (FACS) ve 16S rRNA gen tabanlı sucul ortamlarda aşağılayıcı belirli bir DOC bileşik yetenekli bacterioplankton kültür-bağımsız bir kimlik sağlar moleküler analizler. Üç kat bacterioplankton microcosms BrdU ve bir model DOC bileşik (DOC değişiklikler), ya da sadece BrdU (no ek kontrol) almak için ayarlanır. Yeni sentezlenmiş bakteriyel DNA ve BrdU-işaretli DNA timidin BrdU yerine pozisyonları kolayca immunodetected 1,2 olabilir. Katma DOC bileşik seçici aktive olması beklenen ve bu nedenle hiçbir BrdU esas DOC değişiklikler ve hücre dışı duyarlı hücreler daha yüksek seviyelerde (HI hücreleri) vardır kullanabilmek 24 saat inkübasyon, bacterioplankton sayesinde Ayrıca kontroller (düşük BrdU dahil hücreleri LI hücreleri). Floresan İmmuno sonra, HI hücreleri ayırt edilirler ve fiziksel floresan aktive hücre sıralama (FACS) 3 LI hücreleri ayrılır. Sıralama DOC-duyarlı hücreleri (HI hücreleri) DNA ayıklanır ve taksonomik rRNA gen-temelli analiz PCR, klon kütüphane yapım ve sıralama da dahil olmak üzere sonraki 16S ile tanımlanır.

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

Çevre bacterioplankton bir model ile çözünmüş organik karbon (DOC) bileşik ve bir DNA etiketleme reaktif bromodeoxyuridine (BrdU) inkübe edilir. Daha sonra floresan aktif hücre sıralama (FACS) kullanarak kendi yükseltilmiş BrdU dahil dayalı toplu topluluk, DOC-aşağılayıcı hücreleri ayrılır. Bu hücreler daha sonra sonraki moleküler analizleri ile tespit edilir.

Bromodeoxyuridine (BrdU) Etiketleme ve Çözünmüş Organik Kültür bağımsız Kimlik sonraki Floresan Aktive Hücre Sıralama Karbon-aşağılayıcı Bacterioplankton

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

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

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