响应面法优化海洋沉积物微生物总DNA提取方法  

Optimization of a Marine Sediment Microbial Total DNA Extraction Methods by Response Surface Methodology

作　　者：郭瑞[1] 赵新强[1] 于翠芳[1] 张名爱[1] 程凡升[1] 

机构地区：[1]青岛农业大学食品科学与工程学院,山东青岛266109

出　　处：《青岛农业大学学报（自然科学版）》2016年第3期197-202,213,共7页Journal of Qingdao Agricultural University(Natural Science)

基　　金：国家自然基金(No.31301438);山东省优秀中青年科学家奖励基金(No.BS2013SW034)

摘　　要：海洋沉积物中的微生物是自然界生态系统中不可或缺的组分,在维持环境与海洋生态平衡方面发挥着重要作用。在海洋沉积物微生物的研究中,细胞裂解率低、片段不够完整等问题导致无法高效地提取微生物的总DNA片段。本实验采用响应面分析法对CTAB-SDS-冻融DNA提取法进行优化。根据单因素的实验结果,选取溶菌酶含量、65℃水浴时间、蛋白酶K含量作为影响因子,利用Box-Behnken进行实验设计,将DNA浓度作为响应值,进行DNA提取方法的响应面优化实验。结果发现:当溶菌酶含量为1.13mg/mL、65℃水浴时间为100min、蛋白酶K含量为0.21mg/mL时,DNA提取效果达到最好。验证实验表明:优化后提取DNA的实际值与理论值相近,具有可行性,优化后提取的DNA片段在纯度、浓度方面高于优化之前。The microorganism in the marine sediment is an important member of the nature ecosystem, and plays a vital role in maintening the marine environment and the ecology balance. However, the low cell ly- sis, incomplete fragments and other issues, result in unefficient extraction of total DNA fragments of mi- crobes. This experiment optimized the method of CTAB--SDS--thaw DNA extraction by response surface analysis method. Based on the single factor experiment, the content of lysozyme, the time of 65 ℃ water bath, the content of proteinase K were selected as the influencing factors. The experiments are designed by Box--Behnken. DNA yield was selected as response values for DNA extraction optimization experiments. The results showed that the optimum lysis conditions for soybean dregs were as follows： the content of ly- sozyme was 1.13 mg/mL, the time of 65℃ water bath was 100 min, the content of proteinase K was 0.21 mg/mL. Proven experiments showed that predicted value of extracted DNA and the actual value are simi- lar. The purity and yield of DNA extracted by optimized method are better than before.

关 键 词：响应面法 海洋沉积物微生物 DNA提取 

分 类 号：Q812[生物学—生物工程]