Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies.
A rapid method for the extraction and purification of DNA from environmental samples for molecular cloning applications was developed. The indigenous cells from plant debris, organic materials, sediments, and soils were lysed directly by using DAS-IZ solution and the nucleic acids were precipitated with isopropanol. A simple purification step using DAS-IIZ solution without binding matrix produced highly pure, colorless and undegraded DNA with molecular weight of more than 20 kb. The superiority of this method was tested for wide applications in molecular cloning, i.e., construction of genomic library by using Lambda DASHII Vector and GigapackIII XL, plasmid library, cloning of gene encoding protease, and molecular microbial diversity analysis. An additional advantage of this method is that only 0.1 g of sample is required, if analysis of many samples in short time should be done. To extract large amounts of environmental DNA for molecular cloning lasts only 30 min and to purify it less than 1 h.
Santosa
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DA
Department of Soil Science, Faculty of Agriculture, Bogor Agricultural University, and Indonesian Center for Biodiversity and Biotechnology (ICBB), R.E. Martadinata 8, Bogor 16162, Indonesia. dsantosa@indo.net.id
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D004777:Environment; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D008967:Molecular Biology / Q000379:methods; D010957:Plasmids / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods; D012988:Soil Microbiology; D013997:Time Factors
D004247:DNA