DNA stable-isotope probing

Neufeld, Josh D, Vohra, Jyotsna, Dumont, Marc G, Lueders, Tillmann, Manefield, Mike, Friedrich, Michael W and Murrell, J Colin (2007) DNA stable-isotope probing. Nature Protocols, 2 (4). pp. 860-866.

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Abstract

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.

Item Type: Article
Uncontrolled Keywords: carbon isotopes,centrifugation, density gradient,dna,dna, bacterial,reverse transcriptase polymerase chain reaction
Faculty \ School: Faculty of Science > School of Environmental Sciences
Depositing User: Pure Connector
Date Deposited: 10 Feb 2014 15:24
Last Modified: 24 Jul 2019 19:46
URI: https://ueaeprints.uea.ac.uk/id/eprint/47524
DOI: 10.1038/nprot.2007.109

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