Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 May;74(10):3302-5.
doi: 10.1128/AEM.02630-07. Epub 2008 Mar 21.

Recovery, purification, and cloning of high-molecular-weight DNA from soil microorganisms

Mark R Liles  1 , Lynn L Williamson, Jitsupang Rodbumrer, Vigdis Torsvik, Robert M Goodman, Jo Handelsman
Affiliations

Recovery, purification, and cloning of high-molecular-weight DNA from soil microorganisms

Mark R Liles et al. Appl Environ Microbiol. 2008 May.

Abstract

We describe here an improved method for isolating, purifying, and cloning DNA from diverse soil microbiota. Soil microorganisms were extracted from soils and embedded and lysed within an agarose plug. Nucleases that copurified with the metagenomic DNA were removed by incubating plugs with a high-salt and -formamide solution. This method was used to construct large-insert soil metagenomic libraries.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Flow chart for the recovery, purification, and cloning of HMW metagenomic DNA from soil microorganisms.
FIG. 2.
FIG. 2.
Formamide treatment of soil metagenomic DNA prevents DNA degradation. Lane 1, 1-kb DNA ladder (Promega); lane 2, MidRange II PFGE marker (New England BioLabs); lane 3, BCEF soil DNA plus formamide and salt treatment; lane 4, BCEF soil DNA plus formamide and salt treatment plus Sau3AI (10 U); lane 5, BCEF soil DNA; lane 6, BCEF soil DNA plus Sau3AI (10 U). PFGE conditions (1- to 3-s switch time) were selected to achieve compression of HMW DNA of >300 kb in order to visualize faint amounts of non-formamide-treated DNA.
See this image and copyright information in PMC

References

    1. Bakken, L. R., and V. Lindahl. 1995. Recovery of bacterial cells from soil, p. 9-27. In J. D. Van Elsas and J. T. Trevors (ed.), Nucleic acids in the environment: methods and applications. Springer Verlag, Berlin, Germany.
    1. Berry, A. E., C. Chiocchini, T. Selby, M. Sosio, and E. M. H. Wellington. 2003. Isolation of high molecular weight DNA from soil for cloning into BAC vectors. FEMS Microbiol. Lett. 223:15-20. - PubMed
    1. Bintrim, S. B., T. J. Donohue, J. Handelsman, G. P. Roberts, and R. M. Goodman. 1997. Molecular phylogeny of Archaea from soil. Proc. Natl. Acad. Sci. USA 94:277-282. - PMC - PubMed
    1. Courtois, S., C. M. Cappellano, M. Ball, F. Francou, P. Normand, G. Helynck, A. Martinez, S. J. Kolvek, J. Hopke, M. S. Osburne, P. R. August, R. Nalin, M. Guerineau, P. Jeannin, P. Simonet, and J. Pernodet. 2003. Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products. Appl. Environ. Microbiol. 69:49-55. - PMC - PubMed
    1. Faegri, A., V. L. Torsvik, and J. Goksoyr. 1977. Bacterial and fungal activities in soil: separation of bacteria and fungi by a rapid fractionated centrifugation technique. Soil Biol. Biochem. 9:105-112.

MeSH terms

LinkOut - more resources