药用百合DNA提取和RAPD条件的优化

DNA extraction from and optimization of RAPD reaction system for medical lily

目的确定药用百合最佳随机扩增多态DNA(RAPD)分析方法。方法以药用百合新鲜鳞叶为材料,研究DNA的提取方法,并对影响RAPD反应的各因素进行优化。结果药用百合RAPD反应体系及程序为:在40μL反应体系中,含40 ng模板DNA,0.3μmol/L随机引物,0.2 mmol/L dNTPs,1.7 mmol/L Mg2+,3μL 10×PCRBuffer,1.5 U Taq酶;扩增程序为:第一步94℃预变性2 min,第二步94℃变性1 min,第三步36℃退火45 s,第四步72℃延伸90 s,返回至第二步重复40个循环,第六步72℃延伸10 min,最后4℃保存以备电泳。结论建立了药用百合RAPD的优化反应体系及程序。

Objective To confine the best analysis method by RAPD of the medical lily. Methods DNA of medical lily was extracted from the fresh scale leaves and discussed the RAPD conditions. Results The system and program of RAPD for lily were as follows： The reactions were performed in a volume of 40 μL containing 40 ng of DNA, 0.3 μmol/L primer, 0.2 mmol/L dNTPs, 1.7 mmol/L Mg^2＋, 3 μL 10×PCR Buffer, 1.5 U Taq enzyme; Thermal cycling was programmed by 1 cycle for 2 rain at 94 ℃ , followed by 40 cycles for 1rain at 94 ℃, 45 sec at 36 ℃, and 90 sec at 72 ℃ and finally, by 1 cycle for 10 rain at 72 ℃, storied at 4 ℃. Conclusion The optimized reaction system and program of RAPD for medical lily has established.