DNA stable-isotope probing
DNA stable-isotope probing
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.
860-866
Neufeld, Josh D.
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Vohra, Jyotsna
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Dumont, Marc G.
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Lueders, Tillmann
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Manefield, Mike
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Friedrich, Michael W.
da3780c1-46aa-4451-b9ce-19b69ce366ef
Murrell, J. Colin
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12 April 2007
Neufeld, Josh D.
97a99cce-a614-441b-ab85-dbae37e3c4ba
Vohra, Jyotsna
95a649a5-9049-4a02-ac5c-f48a714eb10b
Dumont, Marc G.
afd9f08f-bdbb-4cee-b792-1a7f000ee511
Lueders, Tillmann
5e98402c-730c-4662-909b-8b8c93bb16a4
Manefield, Mike
d778947a-0610-44bf-a8c5-67809bced6a1
Friedrich, Michael W.
da3780c1-46aa-4451-b9ce-19b69ce366ef
Murrell, J. Colin
244a92ff-dbe1-41cf-9e65-baacbc4a90cf
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), .
(doi:10.1038/nprot.2007.109).
(PMID:17446886)
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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Published date: 12 April 2007
Organisations:
Environmental
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Local EPrints ID: 387949
URI: http://eprints.soton.ac.uk/id/eprint/387949
ISSN: 1754-2189
PURE UUID: c0cb6383-0098-4a98-85f8-6722a2c9d8fc
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Date deposited: 13 Jul 2016 15:53
Last modified: 15 Mar 2024 03:53
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Author:
Josh D. Neufeld
Author:
Jyotsna Vohra
Author:
Tillmann Lueders
Author:
Mike Manefield
Author:
Michael W. Friedrich
Author:
J. Colin Murrell
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