大白菜SRAP反应体系的建立与优化

以大白菜基因组DNA为模板,通过正交试验设计,从Mgz^2＋、Taq酶、dNTPs、引物、模板5种因素4个水平对大白菜SRAP（Sequence-related amplified polymorphism,序列相关扩增多态性）反应体系进行优化,建立了适合于大白菜的SRAP-PCR优化反应体系,该体系为25 μL：Mg^2＋浓度为3.0 mmol/L,dNTPs为0.2 mmol/L,Taq酶1.5 U,引物为0.2 μmol/L,模板DNA73.2 ng.PCR反应程序为：94 ℃预变性5 min;94 ℃变性1 min,35 ℃复性1 min,72 ℃延伸1 min,5个循环;94 ℃变性1 min,50 ℃复性1 min,72 ℃延伸1 min,35个循环,72 ℃延伸10 min.

Optimization of SRAP Reaction System in Chinese Cabbage(Brassica campestris syn. rapa L. ssp. Pekinensis(Lour) olsson)

Using Chinese Cabbage (Brassica campestris syn.rapa L.ssp.Pekinensis (Lour) olsson) genome DNA as template,the major components of SRAP,such as concent rations of Mg²⁺,dNT Ps,Taq DNA polymerase,primers and template,were optimized in this study by orthogonal design in five factors four levels respectively.The results showed that the optimum SRAP reaction system includes Mg²⁺ 3.0 m mol/L,dNTPs 0.2 m mol/L,DNA template 73.2 ng,Taq DNA poly merase 1.5 U and primer 0.2 mol/L in the 25 L volume reaction.The most suitable protocol was initially denaturing at 94 for 5 min,then pre-amplifying at 94 1 min,35 1min and 72 1 min for five cycles,finally amplifying for 35 cycles when the annealing temperature was adjusted to 50.