摘要
PEG能够提高连接酶连接效率,但是对转化率的影响被忽视,特别是基因工程操作进入到精细定量阶段。以BL21(DE3)为宿主细胞,探讨32%PEG200、75℃30min处理对p ET20b质粒转化率的影响。以不经任何处理的1 ng p ET20b转化率100%作为对照,高温处理后转化率为82.3%,PEG处理后转化率为8%,PEG且高温处理后转化率为0%,利用PCR产物回收试剂盒去除PEG可恢复15.3%转化率,说明75℃高温处理对转化率影响很小,PEG则严重影响转化率,PEG且75℃高温处理导致转化失败。通过研究发现PEG严重影响转化率,提出部分恢复转化率的解决方案,并探讨了PEG影响转化率的机理。
PEG could enhance ligation efficiency, but its influence on transformation of plasmid into E. coli is neglected, especially for exact genetic engineering. Treatment effects of 32% PEG200 or/and 75？ C 30 min on the transformation of pET20b into BI21 （DE3） were investigated, because all genes were ligated in vectors to form complete plasmid. Transformation efficiency was set as 100% for 1 ng pET20b without any treatment. Transformation effieiencies were 82.3%, 8%, and 0%, respectively, for treatment with high -temperature, PEG, and PEG with high - temperature. After recovery of pET20b by DNA Clean kit, transformation efficiency was rescued to 15.3%. Therefore, PEG was found to reduce transformation efficiency, which could be partly rescued by using DNA Clean kit. A mechanism was proposed for the transformation reduction of PEG.
出处
《淮阴工学院学报》
CAS
2016年第3期30-33,共4页
Journal of Huaiyin Institute of Technology
基金
国家自然科学基金项目(31371831)
关键词
PEG
转化率
机理
PEG
transformation efficiency
mechanism
