丝瓜ISSR-PCR反应体系的优化

Optimization of ISSR-PCR reaction system for Luffa

以丝瓜为研究材料,通过单因素试验结合正交设计研究ISSR-PCR反应的影响因素,建立反应体系。结果表明:在20μL的反应体系中,含2.0μL 10×buffer,1.5 UTaqDNA聚合酶,0.25 mmol·L-1dNTPs,0.35μmol·L-1引物,30 ng模板DNA,为丝瓜ISSR-PCR分析的最佳反应体系。引物CW32505的最佳退火温度为54.1℃。

To differentiate various luffa populations and for genetic relationship analysis among them,their tender leaves were used in this experiment to establish the reaction system.Factors affecting the ISSR-PCR amplification were selected and optimized through the combination of a single factor and orthogonal design experiment.A stable and reproducible reaction system suitable for ISSR-PCR analysis for luffa containing 2.0 μL 10 × buffer,1.5 U TaqDNA polymerase,0.25 mmol·L-1 dNTPs,0.35 μmol·L-1 primer and 30 ng template DNA per 20 μL was established.And,the optimized annealing temperature was found to be 54.1℃ for the primer（CA）8A.