Modified method of DNA isolation and optimization of PCR protocol for RAPD analysis of endangered medicinal plant Nardostachys jatamansi D C

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Uma M. Singh, Dinesh Yadav, M. K. Tripathi & Manoj K. Yadav

Department of Agriculture Biotechnology S.V.B.P. University of Agriculture and Technology,

Meerut (U.P.), India

Department of Biotechnology,  DDU Gorakhpur University, Gorakhpur (U.P.) India

Soybean Biochemistry Lab Central Institute of Agriculture Engineering, Bhopal, M.P. India

 An important but limiting step in any molecular biological work is the reliable method of DNA isolation and suitable protocol for PCR analysis. Due to abundance of phenols, polysaccharides, terpenoids and other secondary metabolites in the rhizome of Jatamansi, it is a major problem to get high quality DNA. To isolate quality DNA for PCR analysis modified protocol developed in our lab to overcome such perilous problems. We tried to optimize the concentration of PVP, β-mercaptoethanol and NaCl. Upon gel documentation of isolated DNA by modified method evinced single discrete band of genomic DNA and yielded significantly superior, 30-50 µg/g DNA from dry rhizome. The composition of PCR master mix was also modified for RAPD analysis which includes changing the concentration of MgCl2 and observed agood amplification. Reproducible amplifiable products were observed in all accessions.

 

Key words: Dry rhizome, DNA extraction, RAPD, Phenols, Polysaccharides.

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