大字杜鹃RAPD反应体系的优化

Optimization on RAPD reaction system of Rhododendron schlippenbachin Maxim

以大字杜鹃为材料,以改进的CTAB法提取大字杜鹃叶片总DNA,分别就模板DNA浓度,引物浓度,DNTP浓度,Taq DNA聚合酶量及镁离子浓度对大字杜鹃RAPD反应结果的影响进行研究.通过对各因子的组合比较,建立了大字杜鹃RAPD反应的优化体系,即总反应体积为25μL,其中包括10×Taq酶配套缓冲液2.5μL,模板DNA浓度50 mg/L,引物浓度0.5μmol/L,dNTP浓度0.15 mmol/L,Taq DNA聚合酶1 U,Mg2+浓度2.0 mmol/L,其余用重蒸馏水补足.扩增程序为94℃预变性5 min,94℃变性1 min,38℃退火40 s,72℃延伸2min,共41个循环,72℃延伸7 min.

The genomic DNA was extracted from Rhododendron schlippenbachin Maxim . by means of advanced CTAB method. Studied the influences of the concentration of template DNA, primer, dNTP, TaqDNA polymerase and Mg^2＋ on RAPD analysis procedure. Astablished the optimization system of RAPD on the base of the comparation among various influence factor groups：total volume 25μL, comprising of 10 × Taq buffer（2.5 μL）, template DNA concentration 50 mg/L, primer concentration 0. 5 μmol/L, dNTP 0. 15 mmol/L, TaqDNA polymerase 1 U, Mg^2＋concentration 2.0 mmol/L and the supplementary ddH20. The extended program was： 94℃ predenaturation 5 rain, 94 ℃denaturation 1 rain, 38℃annealing 40 s, 72℃ extension 2 rain, 41 cycles in all, 72 ℃ extension 7 min finally.