产品详情
pKillerRed-Mem细胞膜定位质粒
基本信息
启动子: CMV promoter
复制子: Ori,F1 ori,SV40 ori
终止子: SV40 poly(A) signal
质粒分类: 哺乳细胞质粒;哺乳报告质粒;荧光定位质粒
质粒大小: 4787bp
质粒标签: C-mem,C-KillerRed
原核抗性: 卡那霉素Kan
筛选标记: 新霉素Neo/G418
克隆菌株: 大肠杆菌DH5α
培养条件: 37℃,有氧,LB
表达宿主: 293T等哺乳细胞
质粒简介
pKillerRed-mem质粒是一个哺乳细胞红色荧光定位于细胞膜的质粒。pKillerRed-mem is a mammalian expression vector encoding membrane-targeted KillerRed. KillerRed localized on cellular membrane can be used for effective light-induced cell killing.KillerRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al. 1996]. Membrane localization signal (MLS) of neuromodulin is linked to the KillerRed N-terminus. The MLS (Nterminal 20 amino acid residues of neuromodulin) contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets KillerRed to cellular membranes [Skene and Virág 1989]. pKillerRed-mem vector can be used as a source of MLS-KillerRed hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3’-end of the reporter mRNA. SV40 early promoter (PSV40) provides neomycin resistance gene (Neor ) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr ) in E. coli. Kanr /Neor gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.pKillerRed-mem vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of memrane-targeted KillerRed in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman 1985].Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.
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。