Extraction of high molecular weight genomic DNA from soils and sediments.
The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (beta-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.
Lee
Sangwon
S
Department of Microbiology and Immunology, University of British Columbia, Canada. sangwon.ubc@gmail.com
Hallam
Steven J
SJ
D002499:Centrifugation, Density Gradient / Q000379:methods; D005591:Chemical Fractionation / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D019015:Geologic Sediments / Q000032:analysis*; D054892:Metagenome / Q000235:genetics*; D008970:Molecular Weight; D012987:Soil / Q000032:analysis*; D012988:Soil Microbiology; D052616:Solid Phase Extraction
D012987:Soil; D004247:DNA