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pDsRed2-ER内质网定位质粒
基本信息
启动子: CMV promoter
复制子: pUC ori,f1 ori
终止子: SV40 poly(A) signal
质粒分类: 哺乳系列质粒;哺乳荧光质粒;哺乳红色质粒
质粒大小: 4757bp
质粒标签: N-DsRed2
原核抗性: 卡那霉素Kan(50μg/ml)
筛选标记: 新霉素Neo/G418
克隆菌株: DH5α等大肠杆菌
培养条件: 37℃,有氧 LB
表达宿主: 293T等哺乳细胞
培养条件: 无须诱导,瞬时表达
诱导方式: 无需诱导,瞬时表达
5'测序引物: CMV-F(CGCAAATGGGCGGTAGGCGTG)
3'测序引物: SV40-polyA-R(GAAATTTGTGATGCTATTGC)
备注: 哺乳细胞内质网标记质粒
质粒简介
pDsRed2-ER是一种哺乳动物表达载体,用于标记活细胞内质网。DsRed2基因是按人的密码子优化过的野生型DsRed变体,可以更快的成熟和较低的非特异性聚集。
The vector encodes a fusion consisting of Discosoma sp. red fluorescent protein; the endoplasmic reticulum (ER) targeting sequence of calreticulin , fused to the 5' end of DsRed2; and the ER retention sequence, KDEL, fused to the 3' end of DsRed2. DsRed2 is a human codon-optimized variant of wild-type DsRed that has been engineered for faster maturation and lower non-specific aggregation.To drive expression of DsRed2, this vector contains the immediate early promoter of cytomegalovirus (PCMV IE). SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3'-end of the DsRed2 mRNA transcript. The vector also contains an SV40 origin for replication in any mammalian cell line that expresses the SV40 Tantigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassette—consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK poly A) gene— allows stably transfected eukaryotic cells to be selected using G418 . A bacterial promoter (P) upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli.pDsRed2-ER can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 .
质粒图谱
pDsRed2-ER内质网定位质粒使用说明:
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。
pDsRed2-ER内质网定位质粒注意事项:
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。