title: An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries.
abstract: An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification of DNA while minimizing the loss of DNA with respect to other available protocols for metagenomic DNA extraction. Moreover, the purity levels are significant, which are difficult to attain with any of the methods reported in the literature for DNA extraction from soils. The DNA thus extracted was free from humic substances and, therefore, could be processed for restriction digestion, PCR amplification as well as for the construction of metagenomic libraries.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110 021, India.
authors.forename: Digvijay
authors.initials: D
authors.lastname: Verma
authors.forename: T
authors.initials: T
authors.lastname: Satyanarayana
mesh_terms: D019840:2-Propanol / Q000737:chemistry; D001105:Archaea; D001419:Bacteria; D002457:Cell Extracts; D002606:Charcoal / Q000737:chemistry; D019641:DNA, Archaeal / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis*; D004271:DNA, Fungal / Q000032:analysis*; D021901:DNA, Intergenic / Q000032:analysis; D005658:Fungi; D015698:Genomic Library; D054892:Metagenome; D056186:Metagenomics / Q000379:methods*; D059013:Microbial Consortia; D011092:Polyethylene Glycols / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011205:Povidone / Q000031:analogs & derivatives / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012988:Soil Microbiology; D014990:Xylans / Q000378:metabolism
chemical_list: D002457:Cell Extracts; D019641:DNA, Archaeal; D004269:DNA, Bacterial; D004271:DNA, Fungal; D021901:DNA, Intergenic; D012336:RNA, Ribosomal, 16S; D014990:Xylans; D002606:Charcoal; C077842:polyvinylpolypyrrolidone; D011092:Polyethylene Glycols; D011205:Povidone; D019840:2-Propanol