摘要
以假臭草叶片为材料,对影响其随机扩增多态DNA(RAPD)反应的各因素进行优化,建立了假臭草RAPD的优化反应体系和程序,即在10μL反应体系中,5ng(/10μL)模板DNA,1.0μmol/L随机引物F15,150μmol/LdNTPs,2.0mmol/LMg2+,1.0UTaqDNA聚合酶;扩增程序为95℃预变性4min,95℃变性40s,36℃退火40s,72℃延伸1min,10个循环,后94℃变性30s,35℃退火30s,72℃延伸1min,35个循环,72℃延伸5min,4℃保温。
The genomic DNA of Praxelis clematidea was employed to establish optimization of RAPD. The RAPD system for Praxelis clematidea was optimized including program, template, primer, dNTPs, Taq polymerase and Mg^2＋. Through ladder experiments, the reliable RAPD analysis system was established. The total reaction volume was 10 μL and reaction mixture consisted of template DNA, dNTPs, Mg^2＋, random primer and Taq DNA polymerase. RAPD program was 4 min at 95℃ for predenaturation, then 10 cycles of 40 s at 94℃ for denaturation, of 40 s at 36℃ for annealing, of 1 min at 72℃ for extension; then 35 cycles of 30 s at 94℃ for denaturation, of 30 s at 35℃ for annealing, of 1 min at 72℃ for extension, finally extension at 72℃ for 5 min.
出处
《热带农业科学》
2010年第5期21-25,共5页
Chinese Journal of Tropical Agriculture
基金
国家自然科学基金项目(No.30960088)
关键词
假臭草
DNA提取
RAPD
体系优化
Praxelis clematidea
genomic DNA extraction
RAPD
optimization
