桢楠DNA提取和RAPD条件的优化

DNA Extraction and Optimization of RAPD Reaction System for Phoebe zhennan

以桢楠(Phoebe zhennan)新鲜叶片、硅胶干燥叶片为材料,研究了DNA的提取方法,并对影响RAPD反应的各因素进行了优化。得到了桢楠RAPD的优化反应体系及程序,适合桢楠RAPD反应的体系总体积为25μL,DNA模板2μL,Taq DNA聚合酶用量0.3μL,引物用量1μL,Mg2+用量5μL,dNTPs用量0.5μL,ddH2O16.2μL;适合桢楠RAPD扩增的程序是94℃预变性3 min,94℃变性1min,36℃复性1 min,72℃延伸2 min,42个循环,最后72℃延伸10 min,产物于4℃保存,同时结果表明硅胶保存的样品完全可以与传统的新鲜叶片得到同样的PCR扩增结果,完全可以满足研究需要。

DNA of Phoebe zhennan was extracted from the fresh leaves and silica gel dried leaves,and the various factors affecting RAPD reaction was optimized.The optimized reaction system and programme of RAPD for Phoebe zhennana were as follows：each 25 μL amplification reaction solution suited for Phoebe zhennana RAPD was consisted of 2μL DNA,0.3 μLTaq DNA polymerase,1 μL primer,5 μL MgCl2,0.5 μL dNTPs and 16.2 μL ddH2O.The PCR amplification program suited for Phoebe zhennan RAPD were pre-denaturing at 94 ℃ for 3 min,followed by denaturing at 94 ℃ for 1 min,annealing 36 ℃ for 1 min,extending 72 ℃ for 2 min and cycling 42 times,and lastly,extending 72 ℃ for 10 min and storing at 4 ℃.The results showed that same results of PCR amplification could be got from the fresh leaves and silica gel dried leaves and could meet the need for researches.