Optimization of Melia azedarach RAPD Reaction System

Taking genomic DNA extracted from Melia azedarach foliage as experimental material,a RAPD analysis system was established by means of single factor multi-level gradient experiment to select suitable concentration and dosage for template,Mg²⁺,Taq DNA polymerase,dNTPs,and the random primers.The results showed that: Clear electrophoresis bands could be obtained with 60 ng/μL concentration of genomic DNA,3.0 mmol/L of Mg²⁺,0.25 mmol/L of dNTP,0.30 μmol/L of primer,1 U/(20 μL) of Taq DNA polymerase and when the total reaction volume was 20 μL.The optimum RAPD program was 2 min predenaturation at 94 ℃,followed by 38 reaction cycles,8 min extension at 72 ℃ and final storage at 4 ℃.Each reaction cycle included 30 sec of denaturation at 94 ℃,1 min annealing at 37 ℃ and 80 sec extension at 72 ℃.