狭叶坡垒RAPD-PCR反应体系优化

The Optimization of RAPD-PCR System of Hopea chinensis

以濒危物种狭叶坡垒硅胶干燥的叶片为材料,研究其RAPD-PCR体系优化条件。结果表明,优化的狭叶坡垒RAPD-PCR反应体系为:25μL体系中1×PCR buffer,3 mmol/L Mg2+,0.2 mmol/L dNTPs,0.5 U/25μL Taq聚合酶,0.4μmol/L引物,5 ng/μL DNA模板;最佳扩增程序为:94℃预变性5 min;94℃变性1 min,35℃退火1 min,72℃延伸1.5min,35个循环;72℃最后延伸7 min。

To optimize the RAPD reaction condition for Hopea chinensis genomic DNA,the concentrations of MgCl2,dNTPs,Taqpolymerase,primers and template DNA were studied,and the optimal anneal temperature of primer and cycles were determined through gradient PCR.The optimal PCR system for RAPD analysis was 1 × PCR buffer,3 mmol / L MgCl2,0.2 mmol/LdNTPs,0.5 U/25 μL Taqpolymerase,0.4 μmol/L primer,5 ng/μL template DNA in 25 μL reaction solution.And the augmentation procedure was pre-denaturation at 94 ℃ for 5 min,denaturation at 94 ℃ for 1 min,annealing at 35 ℃ for 1 min,extension at 72 ℃ for 1.5 min,reaction with 35 cycles,and extension at 72 ℃ for 7 min.