摘要
建立一整套适用于研究桑天牛线粒体细胞色素氧化酶I(mtDNA CO I)基因的PCR反应体系,研究不同地理分布的桑天牛遗传多样性。通过L_(16)(4~5)正交组合试验和单因素梯度试验对缓冲液(Mg^(2+))、dNTP、随机引物、TaqDNA聚合酶、模板DNA的浓度和退火温度、循环次数等影响PCR扩增的重要因素进行优化。试验结果表明,50μL反应体系及反应程序中各因素优化组合为:缓冲液(含Mg^(2+))0.9mmol/L,dNTP 0.15mmol/L,随机引物0.44μnol/L,TaqDNA聚合酶3.0U,模板DNA75ng;退火温度47℃,循环次数35次。
The establishment of proper PCR reaction system focuses on doing research on the genetic diversity of one kind of main pest called Apriona germari.Several important factors,including buffer（Mg^2＋ ）,dNTP,random primers,Taq polymerase and template DNA were optimized through L16（4^5） orthogonally designed experiment.Annealing temperature and number of amplification cycles were optimized through single factor experiment.It is shown that,by the result of optimization, the optimum PCR reaction system of Apriona mtDNA CO I,a total volume of 50μL PCR reaction mixture,includes 10×buffer（Mg^2＋ ）0.9mmol/L,dNTP0.15 mmol/L,random primers 0.44μmol/L,Taq polymerase 3.0 U,template DNA 75 ng, annealing temperature 47℃and 30 cycles of amplification.
出处
《河北林果研究》
2010年第4期384-388,共5页
Hebei Journal of Forestry and Orchard Research
基金
河北省自然科学基金项目(No.C2010000683)
