北方黑土土壤细菌16S rDNA扩增体系的优化

OPTIMIZATION OF 16S rDNA AMPLIFICATION SYSTEM OF BACTERIA IN BLACK SOIL IN NORTH CHINA

采用降落PCR方法扩增目的基因，设计两次正交试验对PCR反应体系中的各组分浓度进行筛选，同时对退火时间、延伸时间及循环次数进行摸索。结果表明，适合北方黑钙土土壤细菌16SrDNA扩增体系为：在25μl体系中，10×buffer2．5μl，DNA模板20ng，Mg^2＋ 2．5mmol L^-1,dNTPs0．25mmol L^-1，引物0．3μmol L^-1，TaqDNA聚合酶1．5U。降落PCR扩增程序为：95℃，5min；95℃，45s；65～56℃，60s；72℃，90s；每两个循环降低1℃。95℃，45s；55℃，60S；72℃，90s；10个循环。最后72℃，10min。

Abstract Target genes were amplified with the TD PCR method, two orthogonal experiments were designed to screen out optimal concentrations of various components of the PRC reaction system, while to explore annealing time, extending time and cycling frequency. Results show that soil bacteria 16S rDNA amplification system fit for black soil in North China was in 25μl volume, which was composed of 10 × buffer 2.5 μl ,20 ng soil microbial DNA template, Mg^2＋ 2.5 mmol L^-1 ,dNTPs 0.25 mmol L^-1, 0.3 μmol L^-1 primer, and 1.5UTaq enzyme. The TD PCR reaction procedure went like first keeping 95 ℃ for 5 min to make soil microbial DNA denatualized, 95℃ for 45 s more, 65 - 56℃ for 60 s, and then 72℃ for 90 s, and lowering the temporature by 1 ℃ every two cycles; and starting another 10 cycles of 95 ℃ for 45 s, 55 ℃ for 60 s, and 72 ℃ for 90 s, and finally staying at 72℃ for 10 min for extending.