摘要
在利用ISSR技术对黄藤进行分子生物学研究过程中,对影响PCR扩增效果的一些因素,如模板DNA浓度、Mg2+浓度、dNTPs浓度、Taq DNA聚合酶浓度、引物浓度以及退火温度等指标进行筛选和优化,获得用于黄藤ISSR-PCR分析最适宜的PCR反应条件:20μLPCR反应体积中含有1×缓冲液,20 ng模板DNA,2 mmol.L-1MgC l2,100μmol.L-1dNTP,1.5 U TaqDNA聚合酶,0.4μmol.L-1引物,2%甲酰胺,0.5 mmol.L-1亚精胺.扩增程序为:94℃预变性5 m in;再35个循环:94℃30 s,52℃45 s和72℃1.5 m in;最后72℃延伸7 m in.
This paper studied the effecting factors in Daemonorops margaritae （ Hence ） Beccari ISSR analysis. These factors included template DNA concentration, Mg^2＋ concentration, dNTPs concentration, Taq DNA polymerase concentration, primer concentration, and annealing temperature. The optimized conditions for D. margaritae ISSR-PCR reaction were as follows： 1×Taq buffer, 20 ng template DNA, 2 mmol.L^-1 MgC12, 100μmol.L^-1 dNTPs, 1.5 U Taq DNA polymerase, 0. 4μmol. L^-1 primer, 2% formamide and -. 5 mmol.L^-1spenninide. The amplification cycling was 1 cycle at 94 ℃ for 5 min; 35 cycles at 94 ℃ for 30 sec, 52 ℃ for 45 sec, and 72℃ for 1.5 rain respectively ; and 1 cycle at 72 ℃ for 7 rain.
出处
《福建林学院学报》
CSCD
北大核心
2006年第2期152-155,共4页
Journal of Fujian College of Forestry
基金
国家"十五"攻关课题"棕榈藤种质资源培育及利用技术"(2001BA506b04)
关键词
黄藤
ISSR
优化
Daemonorops margaritae
inter-simple sequence repeats
optimization
