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Defining optimum metagenomic procedure for microbial diversity analysis in wheat rhizosphere

Bacteria are an important part of the soil micro flora. Conventional plate or liquid culture often cannot emulate the environmental conditions to which these microorganisms are adapted. Thus, these methods do not necessarily provide comprehensive information on the diversity and biotechnological potential of a soil or water sample. Metagenomics circumvents the unculturability and genomic diversity of most microbes, which is the biggest roadblock to advances in clinical and environmental microbiology. In this study certain soil DNA extraction procedures were studied and evaluated for microbial DNA yield from different regions of North India. Soil samples under wheat crop were collected in sterile condition at a depth of 10-15 cm below the soil surface in the months of Jan-May. All samples were analyzed for their physical properties. DNA was extracted metagenomically by three methodologies after making certain modifications. Purity of isolated DNA was checked by taking O.D at 260 and 280nm. Purest form of DNA was obtained by ASM method as compared to the other two methods. This method was applicable on all soil types and even on old soil samples with fair degree of efficiency. 17.7-40.98 μg of DNA was obtained with a purity ratio of A260/A230 and A260/A280 as 1.456 and 1.680 respectively. Size of DNA obtained was found to be > 23 Kb. DNA obtained by ASM method was used directly for PCR as there were negligible amount of inhibitors left due to addition of CaCl2 .

Author(s): Shagufta Nahid, S. Yasir and Arif Ali

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