Optimization of PCR conditions using RAPD primers for genetic diversity study of java tea (Orthosiphon stamineus Benth)

The random amplified polymorphic DNA (RAPD) technique was used to determine the polymorphism of java tea (Orthosiphon stamineus). The objective of this research was to optimize the PCR conditions by using various RAPD primers to produce clear and pronounced DNA bands. Among the modifications tested in this research are the amount and concentration of magnesium chloride (MgCl₂) used, concentration of PCR buffer, different types of primers and the annealing temperature. The results in this experiment suggested that the best PCR conditions were by using 5µL of 10x PCR buffer, lµL of 1mM MgCl₂, 0.5µL of dNTPs and 0.25µL of 1.25U Taq polyrnerase at the annealing temperature of 60°C. When these conditions were met, both primers C-01 and A-02 produce clear band.