芨芨草基因组DNA提取与RAPD反应体系优化

A Studies on DNA Extraction and Establishmen of RAPD Reaction System in Achnatherum splendens

本文选出适合芨芨草Achnatherum splendens(Trin.)Nevski基因组DNA提取方法,对RAPD反应体系中的一些重要参数进行优化,包括模板浓度、Mg2+、dNTP、引物和Taq酶浓度,建立了适合芨芨草RAPD分析的PCR反应体系。即在12.5uL的反应总体积中Mg2+浓度为1.5mmol.L-1,dNTPs浓度0.2mmol.L-1,Taq酶0.5U,20ng模板DNA,10bp附机引物3.2pmol。反应程序为:前6个循环为95℃变性30s,35℃复性45s,72℃延伸80s,后36个循环为94℃45s,36℃40s,72℃70s,最后72℃延伸10 min。该反应体系具有良好的稳定性和重复性。

Genomic DNA of in A. splendens was successfully extracted by CTAB. Factors influencing RAPD result, including the concentration of DNA template, Mg^2＋ , dNTP, primers and Taq, were studied. An optimul PCR system for RAPD analysis was found： in 12.5 μL reaction solution, contained 10mmol · L^- 1 Tris - HCI （pH8.0）, 50mmol · L^-1 KCl, 1. 50mmol · L^-1 MgCl2, 0.2mmol · L^-1 dNTP, 3.2pmol random primer, 20ng DNA template, 0.5U Taq polymerase. The amplification program was devised： denaturing at 95℃ for 30s, primer annealing at 35℃ for 45s, extension at 72℃ for 80s, 6 cycles; then 94℃ 45s, 36℃ 40s, 72℃ 70s, 36 cycles; and 10min at 72℃ in the final extension. The reaction system was well reproducible and highly rebiable, and it couble be effectively for RAPD analysis in the systematic study in A. splendens.