药用植物白术ISSR-PCR反应体系的正交优化研究

Study on Optimization of ISSR-PCR Reaction System of Atractylodes macrocephala Koidz by Orthogonal Design

采用正交试验设计的方法,对白术ISSR-PCR反应体系进行优化,确立了适合于白术的ISSR-PCR反应的最佳体系,即在25μL反应体系中,10×buffer 2.5μL,Mg2+3 mmol/L,Taq酶0.75U,模板200 ng,dNTP 0.3mmol/L,引物0.5μmol/L,通过梯度PCR试验得到相应引物的最佳退火温度为55.0℃。

The orthogonal design was used to optimize ISSR - PCR reaction system of Atractylodes macrocephala Koidz. A suitable ISSR - PCR system was established and a total volume of 25μL ISSR - PCR system consisted of 10 × buffer 2.5μL, Taq DNA polymerase 0.75U, Mg2 ＋ 3mmol/L, template DNA 200 ng, dNTP 0.3mmol/L, primer 0.5 μmol/L. The optimal annealing temperature of ISSR - PCR for Atractylodes macrocephala Koidz. reaction was proposed by gradient PCR.