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DNA stable-isotope probing

Nature Protocols volume 2, pages 860–866 (2007)Cite this article

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.

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Figure 1: Retrieval of DNA from ultracentrifuge gradients.
Figure 2: Expected results for SIP gradient fractionation.
Figure 3: Expected results for an EtBr-containing gradient.

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Acknowledgements

This work was supported by funding from the Natural Environment Research Council (UK), the Deutsche Forschungsgemeinschaft (SFB 395), the Max Planck Society, the Helmholtz Society and the Natural Sciences and Engineering Research Council (Canada).

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Authors and Affiliations

  1. Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK

    Josh D Neufeld, Jyotsna Vohra, Marc G Dumont & J Colin Murrell

  2. Institute of Groundwater Ecology, GSF—National Research Centre for Environment and Health, Neuherberg, Germany

    Tillmann Lueders

  3. School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, 2052, New South Wales, Australia

    Mike Manefield

  4. Molecular Geobiology Group, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

    Michael W Friedrich

  5. 2007: Department of Biology, Address from July 1, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.,

    Josh D Neufeld

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  1. Josh D Neufeld
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  2. Jyotsna Vohra
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Correspondence to Josh D Neufeld or J Colin Murrell.

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Neufeld, J., Vohra, J., Dumont, M. et al. DNA stable-isotope probing. Nat Protoc 2, 860–866 (2007). https://doi.org/10.1038/nprot.2007.109

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