一种活性污泥总DNA提取方法的优化

对3种活性污泥DNA提取方法———传统蛋白酶K-SDS-氯仿异戊醇法(CPSCI法)、液氮研磨法和脱腐处理法进行了对比,并针对CPSCI法从污泥量、保温时间、裂解方式及沉淀时间等4个方面进行了优化。结果表明,优化的蛋白酶K-氯仿-异戊醇法(OPSCI法)采用污泥量0.10 g、37℃静置10 min,加SDS常温旋涡振荡及异丙醇直接离心等条件可获得长度在23.1 kb左右的DNA,OD260nm/OD280nm为1.86,稀释10倍后即可进行16S rDNA PCR。该方法重复性好,提取得率高,纯度好,操作简便,为常规实验室开展活性污泥微生物多样性研究提供了帮助。

Optimization of DNA extraction from active sludge

Three DNA extraction methods from active sludge,classical proteinase K-SDS-chloroform/isoamyl alcohol method(CPSCI method),liquid nitrogen skiving method and humic acids removing method were compared.CPSCI method was optimized on the quantity of sludge,incubation time,centrifugation time and precipitation time in isopropanol,respectively.The results showed that 23.1 kb genic DNA(OD260nm/OD280nm=1.86) can be obtained by optimized CPSCI method(OPSCI) under the conditions of using about 0.10g sludge,incubating 10 min at 37 ℃,shaking 5min with SDS at room temperature and centrifugating directly after adding isopropanol.The acquired DNA dilution(10 times) can be used for PCR.The OPSCI method introduced in this paper possessed good repeatability,simple operation,and high quality and yield.It was a rapid and advanced method for extracting DNA from active sludge samples in general laboratory.