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pG5Luc哺乳双杂交质粒
基本信息
复制子: pUC ori,f1 ori
终止子: SV40 poly(A) signal
质粒分类: 哺乳细胞,哺乳细胞双杂交载体
质粒大小: 4955bp
原核抗性: Amp
克隆菌株: DH5α
培养条件: 37℃,有氧 LB
表达宿主: 哺乳细胞
诱导方式: 无须诱导,瞬时表达
5'测序引物: Sv40-polyA-R:GAAATTTGTGATGCTATTGC
3'测序引物: 根据序列设计引物
质粒简介
The pG5luc Vector contains five GAL4 binding sites upstream of a minimal TATA box, which in turn is upstream of the firefly luciferase gene. The pGAL4 and pVP16 fusion constructs are transfected along with the pG5luc Vector into mammalian cells. Two to three days after transfection, the cells are lysed, and the amount of Renilla luciferase and firefly luciferase are quantitated using the Dual-Luciferase Reporter Assay System. Interaction between the two test proteins, expressed as GAL4 and VP16 fusion constructs, results in an increase in firefly luciferase expression as compared to the negative controls.
质粒图谱
pG5Luc哺乳双杂交质粒使用说明:
1、收到质粒干粉后请先5000rpm离心1min,再加入20μl无菌水溶解质粒,室温放置1min;
2、从-80℃冰箱中取出相应的感受态,置于冰盒上解冻,并做好标记;
3、取2μl质粒加至100μl感受态中,冰浴30min;
4、42℃热激90s,再冰浴2min;
5、加入900μl无抗的LB液体培养基,180rpm震荡培养45min;
6、6000rpm离心5min,仅留100ul上清混匀菌体沉淀;
7、混匀后的菌液加至对应抗性的LB平板上,倒入适量玻璃珠,涂匀液体;
8、将平板正向培养1h,再倒置培养12h~16h;
9、挑取单克隆菌落至对应抗性的LB液体培养基中,震荡培养12h~16h,根据实验需要提取质粒。
pG5Luc哺乳双杂交质粒注意事项:
1、如果您收到的是甘油菌种,请先四区划线,挑取单克隆培养。
2、如果第二天转化平板长的过多,请将质粒按比例稀释后再转化。