天麻PCR反应体系的建立

Establishment of PCR Reaction System in Gastrodia elata

通过对Mg2+,dNTP,随机引物、模板DNA,TaqDNA聚合酶浓度等反应参数的系统研究,建立了天麻RAPD分析体系。该体系反应总体积20μL,其中MgCl22 5mmol/L,dNTP0 25mmol/L,模板20ng,随机引物0 3μmol/L,Taq酶1 5U.反应程序为94℃预变性5min,94℃变性35s,36℃退火35s,72℃延伸1min,40个循环,最后72℃延伸5min 结果表明该体系具有良好的稳定性和重复性,可应用于天麻的演化、系统分类、品种鉴定等研究。

A RAPD analysis system has been established for Gastrodia elata through the optimization of the concentrations of Mg^(2+), dNTP, random primer, template DNA and Taq DNA Polymerase. The reaction volume was 20μL, containing 2.5mmol/L MgCl2,0.25mmol/L dNTP,20ng template DNA,0.3μmol/L random primer and 1.5U Taq DNA Polymerase. The PCR cycle was designed as following: pre-denaturing at 94℃ for 5min, denaturing at 94℃ for 35s, annealing at 36℃ for 35s, extension at 72℃ for 1min, total 40 cycles, then extension at 72℃ for 5min. The results show that this system is stable and repeatable and can be used in the studies of Gastrodia elata evolution, systematic classification and variety identification.