产品详情
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
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。