瓜蒌ISSR-PCR最佳反应体系的研究  

Study on Optimization for ISSR Reaction System in Trichosanthes kirilowii Maxim

作　　者：王真真[1] 韩琳娜[1] 郭庆梅[1] 张百霞[1] 周凤琴[1] 

机构地区：[1]山东中医药大学药学院,山东济南250355

出　　处：《辽宁中医杂志》2013年第10期2094-2096,共3页Liaoning Journal of Traditional Chinese Medicine

基　　金：国家科技支撑计划(2011BAI06B06);山东省科技发展计划项目(2011GSF11904)

摘　　要：为了得到适合研究瓜蒌农家品种的ISSR-PCR反应条件,本研究采用单因素实验和正交实验,对瓜蒌ISSR反应体系进行了优化,确立了适合瓜蒌的ISSR反应体系:20μL反应体系,含0.5μmol/L引物、2×Master Mix(Taq DNA Polymerase)10μL、35 ng模板DNA。通过性状差异较大样品的单因素试验,确定了能够适合尽量多样品的ISSR最佳反应程序为:94℃预变性5 min;94℃变性30 s,退火1 min,72℃延伸45 s,35个循环;72℃延伸7min,4℃保存。不同的引物所需要的退火温度不尽一致。In this study, optimization for ISSR amplification system Trichosanthes kirilowii Maxim. was determined in two fac- tors （DNA template and primer） at three levels respectively, combined with single factor experiment and orthogonal designed. A suitable ISSR reaction system was established, namely 20 μL reaction system containing 0.5 μmol/L primer, 10 μL of 2 × Master Mix ,35 ng of DNA template. The optimal extended time and annealing temperature for ISSR - PCR reaction were proposed by gra- dient PCR. And the best ISSR PCR procedures included predenaturing at 94 ℃ for 5 rain;30 cycles of denaturing at 94 ℃ for 30s, annealing at 55 ℃for 1 rain and extension at 72 ℃ for 45 s. The amplification was completed by holding the reaction mixture at 72 ＇12 for 7 rain and holding at 4 ℃. The annealing temperature was different according to different primers.

关 键 词：瓜蒌 ISSR 正交设计 单因素 梯度PCR 

分 类 号：R285.5[医药卫生—中药学]