实时荧光定量PCR反应体系的优化研究选编.PDFVIP

134 广东农业科学 2013 年第4 期 实时荧光定量PCR反应体系的优化研究 袁 坤 张 丹, 刘新琼 张向明 中南民族大学生命科学学院生物技术国家民委重点实验室袁湖北 武汉 430074冤 摘要 为了降低实时荧光定量PCR Real-Time Fluorescent Quantitative PCR 袁FQ-PCR冤技术的实验成本袁提高利用效率袁对影 2+ 响FQ-PCR较敏感的因素二甲基亚砜 DMSO冤尧 甘油和Mg 等进行了优化试验遥 结果表明袁10 滋L反应体系的最优组分为院1伊 rTaq Buffer 袁0.16 mmol/L dNTPs袁0.24 滋mol/L Primer 袁2.5 mmol/L Mg2+ 袁5%甘油袁5% DMSO 袁1伊SY BR Green I 袁0.8 U rTaq 酶袁100 ng模板遥该 反应体系为经济型FQ-PCR的广泛应用奠定了基础遥 关键词 实时荧光定量PCR 曰实验技术曰优化 中图分类号 Q781 文献标识码 A 文章编号 1004-874X(2013)04-0134-03 Factor optimization for the reaction of real-time fluorescent quantitative PCR YUAN Kun, ZHANG Dan, LIU Xin-qiong, ZHANG Xiang-ming (Key Biotechnology Laboratory of the State Ethnic A fairs Commiss ion, College of Life Science, South-Central Unive rs ity for Nationalities , W uhan 430074, China) Abstract: Real-time fluorescent quantitative PCR (FQ-PCR) has been widely applied in biological research. To reduce the cost of experiment and increase the used efficiency, the concentrations of the sensitivity factors including DMSO, Glycerin and Mg2 + were further optimized in the FQ-PCR protocol. The results showed that the best optimal system (10 滋L) was as follows: 1伊 rTaq Buffer, 0.16 mmol/L dNTPs, 0.24 滋mol/L Primer, 2.5 mmol/L Mg2 + , 5% Glycerin, 5% DMSO, 1 伊 SYBR Green I, 0.8 U rTaq and 100 ng template. The optimized system was the basis of economical application of FQ-PCR. Key words: real-time fluorescent qua