摘要
以大白菜基因组DNA为模板,通过正交试验设计,从Mg2+、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25μL:Mg2+浓度为3.0 mmol/L,dNTPs为0.2mmol/L,Taq酶1.5 U,引物为0.2μmol/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。
Using Chinese Cabbage （Brassica campestris syn. rapa L. ssp. Pekinensis （Lour） olsson） genome DNA as template, the major components of SRAP, such as concentrations of Mg^2＋ , dNTPs, 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 Mg^2＋ 3.0 mmol/L, dNTPs 0.2 mmol/L, DNA template 73.2 ng,Taq DNA polymerase 1.5 U and primer 0.2 μmol/L in the 25μL volume reaction. The most suitable protocol was initially denaturing at 94℃ for 5 min, then pre-amplifying at 94℃ 1 min, 35℃ lmin and 72℃ 1 min for five cycles, finally amplifying for 35 cycles when the annealing temperature was adjusted to 50℃.
出处
《西北农业学报》
CAS
CSCD
北大核心
2007年第3期119-123,共5页
Acta Agriculturae Boreali-occidentalis Sinica
基金
国家自然科学基金资助项目(编号30270913)
关键词
大白菜
SRAP
优化
正交试验
Chinese cabbage （Brassica campestris syn. rapa L. ssp. Pekinensis （Lour） olsson）
SRAP
Optimization
Orthogonal design
