pCEP4-CAT哺乳表达质粒
基本信息
别名: pCEP4/CAT
启动子: CMV promoter
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: 10891bp
原核抗性: 氨苄青霉素Amp
筛选标记: 潮霉素Hyg
克隆菌株: 大肠杆菌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: Sv40-polyA-R:GAAATTTGTGATGCTATTGC
质粒简介
pCEP4 is an episomal mammalian expression vector that uses thecytomegalovirus (CMV) immediate early enhancer/promoter for high level transcription of recombinant genes inserted into the multiple cloning site. The Epstein-Barr Virus replication origin (oriP) and nuclear antigen (encoded by the EBNA-1 gene) is carried by this plasmid to permit extrachromosomal replication in human, primate, and canine cells. pCEP4 also carries the hygromycin B resistance gene for stable selection in transfected cells.
pCEP4/CAT is provided as a positive control for the relative level of expression of recombinant proteins in a cell line of interest. It expresses the chloramphenicol acetyl transferase (CAT) protein from the CMV enhancer/promoter. Like its parent vector pCEP4, pCEP4/CAT contains the hygromycin B resistance gene for selection.
pCEP4是pCEP4/CAT的对照质粒,插入一个基因CAT.。
质粒图谱
![](http://shqshengbio.com/uploads/allimg/200829/1-200RZ00530641.png)
扩增流程
收到产品后,请先根据产品管壁标签来判断产品形式,并在扩增前准确查找该质粒的抗性、感受态和培养温度。
1、质粒干粉(请务必转化挑单克隆重提后使用)
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;
②取2μl质粒加至100μl 感受态中,冰浴30min后,42℃热激60s,不要搅动,再冰浴2min;
(从第二步开始均要在超净工作台中无菌操作)
③加入500μl无抗的LB液体培养基,180rpm震荡培养45min;
④取10μl、100μl复苏液,并分别涂布至目标质粒抗性的LB平板上;
(可使用本平台的平板涂布专用玻璃珠进行涂布,可以比传统涂布方法获得更多转化子)
⑤将平板正向培养1h,再倒置37度培养16h。如果要求是30度则培养20h;
(如果菌落过多,请将质粒稀释后再转化。如果无菌落,请加入10μl质粒离心全涂转化)
⑥挑取单菌落至LB液体培养基中,加入对应抗生素,220rpm震荡培养14h,根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌:四区划线后挑单菌落培养,酵母菌需要先液体复苏再四区划线,再挑单菌落液体培养。
3、穿刺菌:穿刺接种,液体培养后四区划线,再挑单菌落液体培养。
4、平板:直接挑取单菌落至液体培养基中。
5、液体质粒:单独提取的液体质粒收到后可直接使用。