产品介绍
pE2F-TA-Luc哺乳报告质粒
基本信息
原核抗性: 氨苄青霉素Amp
筛选标记: 荧光素酶Luc
克隆菌株: 大肠杆菌DH5α
培养条件: 37℃
质粒简介
pE2F-TA-Luc单荧光素酶信号通路报告质粒。pE2F-TA-Luc is designed to monitor the induction of E2F-mediated signal transduction pathways. E2F, a major target of the retinoblastoma gene (Rb), is a key regulator of cell-cycle checkpoints in mammalian cells . E2F plays a critical role in stimulating expression of genes encoding growth-promoting proteins, and is involved in regulating the expression of important genes during cell proliferation . The E2F protein forms a heterodimer complex with the DP1 protein, which binds to E2F response elements and initiates transcription of genes necessary for DNA replication. Studies have shown that deregulation of E2F results in a loss of cell-cycle checkpoints—thereby predisposing cells to uncontrolled growth . pE2F-TA-Luc contains four copies of the E2F enhancer element (6), located upstream of the minimal TA promoter, the TATA box from the herpes simplex virus thymidine kinase promoter (PTA). Located downstream of PTA is the firefly luciferase reporter gene (luc). Upon binding of the E2F/DP1 complex to the cis-acting E2F enhancer element, transcription is induced and the reporter gene is activated. The luciferase coding sequence is followed by the SV40 late polyadenylation signal to ensure proper, efficient processing of the luciferase transcript in eukaryotic cells. A synthetic transcription blocker (TB) is located upstream of the cis-acting enhancer element. It is composed of adjacent polyadenylation and transcription pause sites for blocking nonspecific transcription (7). The vector backbone also contains an f1 origin for single-stranded DNA production, a pUC origin of replication, and an ampicillin resistance gene for propagation and selection in E. coli.
pE2F-TA-Luc is designed for monitoring cell-cycle signaling in mammalian cells by assaying for luciferase activity. For example, induction of E2F-mediated signal transduction pathways may be compared across different cell types or cell states by transiently transfecting this vector into appropriate cell lines. After transfection, treat each culture individually with a drug candidate or stimulus of interest, then compare the activation of the E2F response element by assaying for the luciferase reporter gene. Additionally, you can monitor pathway activation by cotransfecting this vector with an expression vector containing a gene of interest. Luciferase is a highly sensitive enzymatic reporter that can be assayed by any standard luciferase-detection method, providing quantitative data on induction levels. pE2F-TA-Luc can be transfected into mammalian cells by any standard method. For selecting stable clones, cotransfect with a vector containing an antibiotic resistance gene, such as neomycin, hygromycin, or puromycin, and selecting resistant clones。
质粒图谱
pE2F-TA-Luc哺乳报告质粒使用说明:
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。
pE2F-TA-Luc哺乳报告质粒注意事项:
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。