摘要
采用正交实验设计,对影响土壤微生物RAPD扩增体系的Mg2+、dNTP浓度及引物浓度进行了研究,同时对退火温度、延伸时间及循环次数进行摸索。结果表明,适宜土壤微生物PCR扩增反应在25μl体积中进行,包括7ng土壤微生物DNA样品、20pm随机引物l、.5uTaq酶、3.0mmol.L-1Mg-CL2和0.2mmol.L-1dNTP。PCR扩增反应进程如下:94℃3min,使土壤DNA变性;然后再进行39个循环,每个循环包括94℃1min,37℃40s,72℃90s,结束后72℃延伸7min。
With orthogonal experiment, this paper studied the effects of the concentrations of Mg^2＋, dNTP and primer,the anneal temperature, and the extending time and cycling times on RAPD of soil microbes. The results showed that the feasible PCR reaction system for soil microbes should be carried in 25 μl volume, which was composed of 7 ng soil microbial DNA template, 20 pm random primer, 1.5 uTaq enzyme, 3.0 mmol· L^-1 Mg-CL2, and 0.2 mmol· L^-1 dNTP, The PCR reaction procedure was firstly, keeping 94 ℃ for 3 min to make soilmicrobial DNA denatualize followed by 39 cycles, each including 94 ℃ for 1 rain, 37 ℃ for 40 s, and 72 ℃ for 1.5 min, and finally, keeping 72 ℃ for 7 min for extending.
出处
《生态学杂志》
CAS
CSCD
北大核心
2005年第8期921-924,共4页
Chinese Journal of Ecology
基金
国家自然科学基金(30230250)
黑龙江省自然科学基金资助项目(C0215)。
