桔梗RAPD反应体系的优化

Optimization of RAPD conditions of Platycodon grandflorus

以通化桔梗为材料,用改进的CTAB法提取桔梗叶片的总DNA,通过对不同镁离子浓度、dNTP浓度、模板DNA含量、引物浓度、DNA聚合酶量条件下的RAPD扩增反应的效果,建立了一个适合桔梗的比较稳定的RAPD反应体系,用于桔梗遗传多样性分析。结果表明,桔梗RAPD扩增反应的最佳体系为:模板DNA20ng,dNTP150μmol/L,引物0.3μmol/L,Mg2+浓度2.0mmol/L,TaqDNA聚合酶1Unit,10×Buff-er2.0μL,PCR反应总体积为20μL。按此优化RAPD条件进行实验,重现性良好。

The total DNA in young leaves of Platycodon grandiflorus from Tonghua was successfully extracted by using the developed CTAB method. The optimum reaction system of RAPD for the P. grandiflorus was established by RAPD analysis of the different Mg^2＋ ,DNA templates,primers and DNA polymerase. The results show that the most optimal reaction system of RAPD is： 20 ng template DNA, dNTP concentration 150 μL,0.3 μmol/L primer, MgCl2 2.0 mmol, 1 U Taq polyrnerase, 10 × Taq polymerase buffer at 2. 0 μL in the 20 μL reaction volume. A high reproducibility was obtained with the optimized experiment condition.