Publication: Isolation and cloning of high-molecular-weight metagenomic DNA from soil microorganisms
2
Issued Date
2009-12-01
Resource Type
ISSN
15596095
Other identifier(s)
2-s2.0-73349107144
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Mahidol University
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SCOPUS
Bibliographic Citation
Cold Spring Harbor Protocols. Vol.4, No.8 (2009)
Suggested Citation
Mark R. Liles, Lynn L. Williamson, Jitsupang Rodbumrer, Vigdis Torsvik, Larissa C. Parsley, Robert M. Goodman, Jo Handelsman Isolation and cloning of high-molecular-weight metagenomic DNA from soil microorganisms. Cold Spring Harbor Protocols. Vol.4, No.8 (2009). doi:10.1101/pdb.prot5271 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/27106
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Title
Isolation and cloning of high-molecular-weight metagenomic DNA from soil microorganisms
Abstract
The successful construction of large-insert community DNA (i.e., metagenomic) libraries from natural environments is dependent on several parameters, including effective cell lysis, DNA purity, and a high transformation efficiency. One problem associated with constructing metagenomic libraries from soil microbes is the co-isolation of contaminants, leading to the degradation of DNA as a result of nuclease activity. Because the isolation of intact genetic pathways from soil microbes is necessary to characterize their genetic and functional diversity, obtaining high-purity, high-molecular-weight (HMW) DNA for library construction is absolutely critical. This protocol describes the steps for the indirect extraction of bacterial DNA from soil, embedding the DNA in an agarose matrix, using a formamide and high-salt treatment to eliminate nucleases, size-selecting DNA by restriction digestion and pulsed-field gel electrophoresis (PFGE), and cloning the HMW DNA into a large-insert vector. The resulting metagenomic libraries contain high-purity, stable, HMW DNA that can be screened for various genetic loci (sequence-based) or phenotypic traits (function-based). Copyright © 2009 by Cold Spring Harbor Laboratory Press.