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An improved electroelution method for separation of DNA from humic substances in marine sediment DNA extracts

  • We present a method for the rapid and simple extraction of DNA from marine sediments using electroelution. It effectively separates DNA from compounds, including humic substances, that interfere with subsequent DNA quantification and amplification. After extraction of the DNA from the sediment into an aqueous solution, the crude sample is encased in 2% agarose gel and exposed to an electrical current, which draws the DNA out of the gel into a centrifugal filter vial. After electroelution, the sample is centrifuged to remove contaminants <= 100 000 Da. Recovery of DNA using this method is quantitative and does not discriminate on the basis of size, as determined using DNA standards and DNA extracts from environmental samples. Amplification of DNA is considerably improved due to removal of PCR inhibitors. For Archaea, only these purified extracts yielded PCR products. This method allows for the use of relatively large volumes of sediment and is particularly useful for sediments containing low biomass such as deeply buried marineWe present a method for the rapid and simple extraction of DNA from marine sediments using electroelution. It effectively separates DNA from compounds, including humic substances, that interfere with subsequent DNA quantification and amplification. After extraction of the DNA from the sediment into an aqueous solution, the crude sample is encased in 2% agarose gel and exposed to an electrical current, which draws the DNA out of the gel into a centrifugal filter vial. After electroelution, the sample is centrifuged to remove contaminants <= 100 000 Da. Recovery of DNA using this method is quantitative and does not discriminate on the basis of size, as determined using DNA standards and DNA extracts from environmental samples. Amplification of DNA is considerably improved due to removal of PCR inhibitors. For Archaea, only these purified extracts yielded PCR products. This method allows for the use of relatively large volumes of sediment and is particularly useful for sediments containing low biomass such as deeply buried marine sediments. It works with both organic-rich and -poor sediment, as well as with sediment where calcium carbonate is abundant and sediment where it is limited; consequently, adjustment of protocols is unnecessary for samples with very different organic and mineral contents.show moreshow less

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Author details:Jens KallmeyerORCiDGND, David C. Smith
URL:http://www3.interscience.wiley.com/cgi-bin/issn?DESCRIPTOR=PRINTISSN&VALUE=0168-6496
DOI:https://doi.org/10.1111/j.1574-6941.2009.00684.x
ISSN:0168-6496
Publication type:Article
Language:English
Year of first publication:2009
Publication year:2009
Release date:2017/03/25
Source:FEMS Microbiology ecology. - ISSN 0168-6496. - 69 (2009), 1, S. 125 - 131
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Geowissenschaften
Peer review:Referiert
Institution name at the time of the publication:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Erd- und Umweltwissenschaften
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