Genomic DNA Extraction and RAPD Protocols for Cymbidium hybridium

Abstract:

SDS, CTAB and modified CTAB methods were used to extract DNA from the leaves of Cymbidium hybridium cv. Hiroshima Golden Cup “Sunny Moon” and cv. Fortissimo “Pianist”. The results showed that modified CTAB method was best for DNA quality and production. The optimal reaction of RAPD in C. hybridium was studied with primer 5'-GGTGCTCCGT-3' for cv. Hiroshima Golden Cup “Sunny Moon”. The optimum concentration of five important components such as Mg²⁺, Taq DNA polymerase, primer, template DNA, and dNTP in 25 μl RAPD reaction system were 2.0 mmol/L, 1.0 U, 0.20 μmol/L, 25 ng, and 0.20 mmol/L, respectively. Modified thermal profile consisted of an initial denaturation step at 94℃ for 4 min, followed by 40 cycles of 94℃ for 30 s, 37℃ for 1 min and 72℃ for 1 min and a final exposure to 72℃ for 7 min.