pIRESneo3哺乳表达质粒

pIRESneo3哺乳表达质粒

基本信息

启动子: CMV,T7

复制子: pUC ori

终止子: SV40 poly(A) signal

质粒分类: 哺乳细胞，蛋白过表达载体

质粒大小: 5.2kb

原核抗性: Amp

筛选标记: Neo

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: CMV-F：CGCAAATGGGCGGTAGGCGTG

3'测序引物: 根据序列设计引物

质粒简介

        pIRESneo3 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA (1–3). After selection with G418, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. To select for cells that express high levels of the gene of interest, the selective pressure for antibiotic resistance was increased by shifting the neomycin phosphotransferase gene downstream to a less optimal position for translation as directed by the IRES sequence (1). By decreasing the level of expression of the antibiotic resistance marker, the selective pressure on the entire expression cassette is increased, resulting in the selection of cells that express the entire transcript, including the gene of interest, at high levels.

质粒图谱

pIRESneo3哺乳表达质粒使用说明：

     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。

pIRESneo3哺乳表达质粒注意事项：

      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。