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Neufeld, J.D.* ; Vohra, J.* ; Dumont, M.G.* ; Lüders, T. ; Manefield, M.* ; Friedrich, M.W.* ; Murrell, J.C.*

DNA stable-isotope probing.

Nat. Protoc. 2, 860-866 (2007)
DOI
Open Access Green as soon as Postprint is submitted to ZB.
Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4–5 days, with much of the time being committed to the ultracentrifugation step.
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Publication type Article: Journal article
Document type Scientific Article
ISSN (print) / ISBN 1754-2189
e-ISSN 1750-2799
Journal Nature Protocols
Quellenangaben Volume: 2, Issue: 4, Pages: 860-866 Article Number: , Supplement: ,
Publisher Nature Publishing Group
Reviewing status Peer reviewed
Institute(s) Institute of Groundwater Ecology (IGOE)