硅胶干燥胡杨叶片DNA提取与RAPD反应体系的建立

以塔里木河流域胡杨(Populus euphratica)硅胶干燥叶片为材料,采用改进CTAB法提取胡杨基因组DNA,得到满足RAPD(Random amplif ied polymorphic DNA),随机扩增多态性DNA分析的胡杨基因组DNA。通过单因素优化试验,得到胡杨RAPD分析的最佳反应体系为:40 ng模板DNA,0.8 U rTaqDNA聚合酶,2.5 mmol/L Mg2+,0.3 mmol/L dNTPs,0.5μmol/L随机引物,1×buffer缓冲液,ddH2O补足至10μL。扩增反应程序为:94℃预变性5 min,94℃变性30 s,38℃复性30 s,72℃延伸120 s,35个循环,最后72℃延伸7 min。

Study on DNA Extraction from Silica Gel Dried Leaves of and Establishment of RAPD Reaction System

The authors describe an improved CTAB method for preparation of DNA from the silica-gel-dried leaves of which taken from Tarim River valley.The purity genetic DNA had been obtained and could be used for RAPD PCR.After the optimization of RAPD reactions,the optimized reaction system and program of RAPD were as follows.The 10 μL PCR reactions contained 40 ng DNA template,0.8 U rTaq DNA polymerase,2.5 mmol/L Mg2+,0.3 mmol/L dNTPs,0.5 μmol/L random primer and 1×buffer solution.The amplification program started with 5 min at 94℃,followed by 35 cycles for 30 s at 94℃,30 s at 38℃,and 120 s at 72℃ and finally by 1 extension step for 10 min at 72℃.