白僵菌RAPD-PCR反应体系优化

为了建立一套适宜于白僵菌(Beauveria bassiana)退化研究的RAPD-PCR反应体系及反应程序,通过采用L16(45)正交试验及退火温度和循环次数的单因素优化对反应体系中的各因素进行优化组合。结果表明:20μL PCR反应体系及反应程序中各因素优化组合为,10×Buffer 2μL,MgCl2(25 mmol/L)2.4μL,4种dNTP(各2.5mmol/L)0.8μL,随机引物(10μmol/L)1.4μL,TaqDNA聚合酶(5 U/μL)0.4μL,模板DNA(10 mg/L)1μL。反应条件为,94℃预变性2 min,94℃变性30 s,38℃退火40 s,72℃延伸1 min,循环次数40次,72℃延伸5 min。

RAPD-PCR Reaction System for Beauveria bassiana

An experiment was conducted to establish an optimal PCR(polymerase chain reaction) reaction system and procedure for the degradation of Beauveria bassiana.Single factor test and L16(45) orthogonal experiment were used to optimize the combination of factors for the reaction system.The optimum factor combination was obtained with 20μL reaction volume containing 2μL 10×Buffer,2.4μL MgCl2(25mmol/L),0.8μL four types of dNTPs(each 2.5mmol/L),1.4μL random primer(10μmol/L),0.4μL Taq polymerase(5U/μL),and 1μL template DNA(10mg/L).Reaction conditions were as follows: predenaturing at 94 degrees C for 2min,followed by 40 cycles of denaturing at 94 degrees C for 30s,annealing at 38 degrees C for 40s,extension at 72 degrees C for 1min,and final extension at 72 degrees C for 5min.