土壤微生物总DNA的V_3可变区PCR反应体系优化

为了优化以土壤微生物DNA为模板的PCR反应条件,采用E.Z.N.A.soil DNA kit试剂盒提取土壤微生物总DNA,对16SrDNA V3可变区的PCR反应体系和反应条件进行优化。主要从DNA模板用量、引物浓度、退火温度、热启动方式4个方面进行筛选试验,最后得出最适宜的土壤DNA扩增体系为:10.5ng模板DNA、5μL 10×buffer、4μL 2.5mmol/L dNTP、10μmol/L引物各1.5μL,2.5UTaq酶,加无菌ddH2O补足至50μL;PCR循环程序为:94℃预变性5min;94℃变性1min,52℃退火1min,72℃延伸70s,29个循环;72℃延伸5min。试验结果表明:选择热启动方式和合适的退火温度是获得高质量PCR产物的关键。

Optimization of V3 Region PCR Reaction System with Soil Microbial Total DNA as Template

The optimization of PCR reaction system and condition for 16S rDNA V3 region was carried out with soil microbial total DNA as template,which was extracted using E.Z.N.A.soil DNA kit.The PCR reaction was optimized from the aspects of DNA template concentration,primer concentration,annealing temperature,and hot-start.According to the results,the most suitable PCR system was established in a 50 μL reaction volume containing 10.5 ng of DNA template,5μL of 10×PCR buffer,4μL of 2.5 mmol/L dNTP,1.5μL of 10μmol/L primers for each,2.5U of Taq DNA polymerase.The reaction was built up using the conditions:94 ℃ for 2 min;29 cycles of 94 ℃ for 60 s,52 ℃ for 60 s,72 ℃ for70 s;72 ℃ for 5min.The results demonstrated that hot-start PCR and suitable annealing temperature were important to get high-quality PCR products.