Rapid DNA Isolation Protocol for ISSR and SSR Markers Analysis in Trans-Himalayan Cabbage Species

Abstract

In the present study, we present a simple one day protocol for DNA extraction from cabbage, with no requirement of RNase and overnight precipitation with isopropanol. Due to high endogenous phenolics and polysaccharides and other substances in plants, it is difficult to isolate DNA with high concentration and optimum ratio. With added protein and RNA contamination, ISSR and SSR based primer amplification pose a challenge for the same. This method involves modified CTAB protocol with fine crushing by liquid nitrogen. The reagents required during the extraction are CTAB, NaCl, EDTA, Tris HCl and Ascorbic acid. The extraction buffer should be freshly prepared on the same day as results indicated low yield with overnight extraction buffer. The yield of DNA isolated varies from 107.2 to 3855 ng/µl with ratio ranging from 1.79 to 1.88. The primers developed for the case study were mainly ISSR (inter Simple Sequence Repeats) and SSR (Simple sequence Repeats) which were first standardized for PCR reaction mixture such as MgCl2 (1.8 mM), primers (0.2µM) and Taq Polymerase (0.3 units) with 20 ng/µl of DNA. The gradient PCR showed the annealing temperature of all the primers ranging from 55 to 64°C. Polymorphism was seen among all the amplified PCR products run on 2% agarose gel electrophoresis.