Optimization on SSR-PCR reaction system of Chinese fir
It's important to optimize the SSR-PCR(Polymerase Chain Reaction)reaction system fiRStly when doing research with SSR markers in Chinese fir.The orthogonal design of L16 (4)5 was used to optimize the SSR-PCR system for Chinese fir with four levels and five factors,namely,Mg2+,dNTPs,primer,DNA polymerase and DNA template.The results of PCR were evaluated by variance and intuitive analysis.Then,we respectively determined the concentration of Mg2+,dNTPs,primer and annealing temperature using single factor experiment design.The results showed that the optimum reaction system was 20 μL reaction solution,containing Mg2+ 1.0mmol/L,dNTPs 0.15 mmol/L,primer 0.25 μmol/L,Taq polymerase 0.05 U/μL,DNA template 30 ng,2 μL 10 × PCR buffer and adding ddH2O to 20 μμL,and the annealing temperature was the 63-53℃.To verify the reaction system,we used 4 pairs of EST-SSR markers to amplify 10 F1 Chinese fir plantlets and their parents,and we got clear and stable bands,which showed that the SSR-PCR system was suitable further SSR research in Chinese fir.
Chinese firmolecular markerSSR-PCRorthogonal experimentPCR system optimization
国家科技期刊平台
NSTL
万方数据
