鳡鱼ISSR-PCR反应体系的正交优化

Optimization for ISSR Reaction System of Yellowcheek Carp Elopichthys bambusa by Orthogonal Design

利用正交试验设计的方法,对鳡鱼ISSR-PCR反应体系中的Mg2+浓度、引物浓度、dNTPs浓度、DNA模板浓度及Taq DNA聚合酶浓度进行优化分析。建立了最佳ISSR-PCR反应体系,即25μL反应体系中含Mg2+2mmol/L、引物0.4μmol/L、dNTPs 0.2mmol/L、模板DNA 100ng、Taq DNA聚合酶1.0U。并在此基础上对退火温度和循环次数进行梯度试验,得到最佳退火温度为52℃,最佳循环次数35次。

The orthogonal design was used to optimize ISSR-PCR system of yellowcheek carp Elopichthys bambusa in five factors（Mg2＋,primer,dNTPs,DNA template and Taq DNA polymerase）.The optimized ISSR-PCR system（25 μL） was established under Mg2＋ 2.0 mmol/L,primer 0.4 μmol/L,dNTPs 0.2 mmol/L,DNA template 100 ng and Taq polymerase 1.0 U.Then,annealing at 52 ℃ and 35 cycles were found to be optimal in the optimized ISSR-PCR system.