摘要
利用磷酸钙转染法将lacZ-pShuttle质粒导入HEK 293T细胞中,实验证明,pH值、DNA纯度、转染后培养时间和甘油休克时间是影响转染效率的重要因素。转染后培养6h进行甘油休克效果最佳,比培养0h直接进行休克转染效率升高584.4%;甘油休克时间为4.5min时转染效果最佳,比不用甘油休克转染效率升高130.8%。通过对影响转染的几个条件进行优化,建立了1种可以高效转染HEK 293T细胞的方法,且简便易行。
The HEK 293T cells were transfected by calcium phosphate method by using lacZ-pShuttle plasmid DNA, which inserted a lacZ reporter gene in the MCS of the pShuttle plasmid. The expression of lacZ gene was measured with 13-galactosidase protein dyed by X-gal, which revealed the efficiency of transfection. The influences of the pH value, the purity of the plasmid DNA, the cultured time before shocking and the glycerol-shock time to the transfection efficiency were evaluated. The results show that, the best cultured time before shocking is 6 hours, which makes the transfection efficiency increase by 584.4% and the best time of glycerol-shock is 4.5 minutes, making the efficiency increase by 130.8 % compared to the negative group. The pH value of 6.96 and the pure plasmid DNA are also a key factor, affecting the efficiency of transfection significantly. A high-efficiency calcium phosphate transfection method is established by optimizing several transfection conditions. Its efficiency is much higher than the methods which have been reported previously, and it is very easy to use and stable.
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2005年第5期807-810,814,共5页
Periodical of Ocean University of China
基金
山东省自然科学基金项目(Y2000C09)资助
关键词
磷酸钙转染
甘油休克
X-gal染色
calcium phosphate transfection
glycerol-shock
X-gal dying
