金龟甲基因组DNA RAPD反应条件的优化

Optimization of scarab genome DNA RAPD analysis conditions

以华北大黑鳃金龟[Holotrichia oblita (Faldermann)]和矮臀鳃金龟(H.ernesti Reitter)为供试材料,对DNA模板、Mg2+、dNTPs浓度、淬火和循环内延伸时间及循环数进行优化.提出了金龟甲RAPD分析的最佳反应体系,即25 μL体系中含DNA模板1.2 ng·μL-1、Mg2+ 2. 5 mmol·L-1、dNTPs 0. 25 mmol·L-1;并确定最适淬火时间为40 s,循环内延伸时间为2 min;最佳循环数为40.

The optimized RAPD analysis conditions which involve concentration of DNA template, Mg^2＋ and dNTPs, time of annealing and extension in 1 recycle, and recycle frequency were investigated using Holotrichia oblita （Faldermann） and H. ernesti Reitter genome DNA as template. The optimized reaction system is that 25 μL total volume containing 1.2 ng·μL^ - 1 template DNA, 2.5 mmol · L^- 1Mg^2 ＋ and 0.25 mmol· L ^- 1 dNTPs, annealing for 40 s and extending for 2 min, respectively and the recycling 40 times.