A Modified Protocol for Microbial DNA Extraction and Purification in Crude Oil-Spilled Marsh Soil

Abstract:

High level of humus existing in oil-spilled marsh soil could co-extract with DNA to inhibit PCR amplification. We established a stable DNA extracting protocol by modifying direct lysis and activated sludge DNA extracting procedures: with 0.1g/ml PVPP in extracting buffers to pretreat soils and three cycles of freeze-thawing treatment added, by employing lysozyme and 15% SDS, through reducing duration of water bath and digesting RNA with Rnase A before phenol/chloroform extraction. With high concentration of DNA, this protocol provided lower OD230/OD260 value and appropriate OD260/OD280 ratio, which both were significantly different from 0.1g/ml PVPP & 1% CTAB method, indicating pure DNA solutions. Additionally, DNA purity was further checked by amplifying V3-V5 regions of 16S rRNA on bacterial DNA with primer pair 341F/929R.PCR products confirmed that crude oil-spilled marsh soil pretreated by PVPP protocol and extracted by modified extracting procedure could clear humus better and obtain good quality of DNA.

• Periodical: Applied Mechanics and Materials (Volume 340)
• Pages: 353-357
• DOI: https://doi.org/10.4028/www.scientific.net/AMM.340.353
• Online since: July 2013
• Authors: Ying Wang, Jiang Feng, Guo Ping Wang, Xian Guo Lv, Xiao Yu Wang
• Keywords: DNA Extraction, DNA Purification, Oil-Spilled Marsh Soil, PCR Checking

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