散斑壳属真菌RAPD-PCR反应条件的优化

Optimization of RAPD-PCR Reaction Conditions for Lophodermium

以分离自四川省二郎山林场云南松上的松针散斑壳菌(Lophodermium pinastri)为材料,提取其总基因组DNA做为RAPD-PCR体系优化的模板。研究了dNTPs、Mg^(2+)、DNA模板、Taq酶、引物等组分对RAPD反应结果的影响,并建立了最佳反应体系:25μL PCR反应体积,10×Taq酶缓冲液2．5μL,1．5 U Taq酶,20 ng DNA模板,0．4 mmol/L dNTPs,4．0 mmol/L Mg^(2+),0．48μmol/L引物,10．2μL ddH_2O。

Genomic DNA of fungus Lophodermium China pinastri was extracted from contaminated Pinus. yunnanensis needles collected from Erlang Mountain, Sichuan province.The component effects of dNTP, Mg^2＋ , DNA templates, Taq polymerase,and primers on RAPD-PCR results were studied and the optimal reaction system was established. The optimal reaction conditions were： 10 × Taq polymerase corresponding buffer, 1.5 U Taq polymerase, 20 ng DNA templates,0.4 mmol/L dNTPs, 4.0 mmol/L Mg^2＋ ,0.48 μmol/L primer,and 10.2 μL ddH2O in total 25 μL reaction volumes.