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首页> 外文期刊>Indian Journal of Ecology >Optimisation of Genomic DNA Extraction and PCR Procedure for Sal (Shorea robusta)
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Optimisation of Genomic DNA Extraction and PCR Procedure for Sal (Shorea robusta)

机译:Optimisation of Genomic DNA Extraction and PCR Procedure for Sal (Shorea robusta)

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摘要

In the present study, leaves of Sal were collected from natural populations to isolate the genomic DNA. Unfortunately, genomic DNA extracted using protocols developed by Agbagwa et al (2012) and Doyle and Doyle (1987) was inadequate and failed to obtain genomic DNA of high quality and quantity. Woody tree species are rich in higher concentrations of polyphenols, polysaccharides, and secondary metabolites and interfere in the genomic DNA isolation process. To obtain the genomic DNA in good quality andquantity, the cetyl-trimethyl ammonium bromide (CTAB) method was modified. Modifications were carried out in the concentration of PVP and 2-mercaptoethanol and the process of utilising these reagents. The quantity of extracted DNA was evaluated througha NanoBio spectrophotometer (Analytical Technologies Ltd.), and the quality was checked by 0.8 agarose gel electrophoresis. The standardised protocol yielded high molecular weight DNA in the range of 830.12 ng jil"1to 1597. 23 ng jil"1 with an average of 1278.28 ngmul~(-1). The absorbance ratio at 260 to 280 nm ranged from 1.81 to 1.90, with an average of 1.84, which confirmed the purity of isolated DNA and indicated the presence of very low levels of protein, RNA, and polysaccharide contaminants. Theextracted DNA was used to optimise the PCR conditions for microsatellite markers and obtain sharp polymorphic bands in an agarose gel.

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