产品介绍
pCDNA3.0哺乳表达质粒
基本信息
启动子: CMV promoter
复制子: ColE1 ori,f1 ori
终止子: bGH poly(A) signal
质粒分类: 哺乳系列质粒;哺乳表达质粒;pCDNA系列质粒
质粒大小: 5446bp
质粒标签: 无
原核抗性: 氨苄青霉素Ampicillin(100μg/ml)
筛选标记: 新霉素Neomycin /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: 37℃,5%CO2
诱导方式: 无需诱导
5'测序引物: pCDNA3.1-F(CTAGAGAACCCACTGCTTAC)
3'测序引物: pCDNA3.1-R(TAGAAGGCACAGTCGAGG)
备注: 常用的哺乳细胞真核表达载体
质粒简介
pCDNA3.0载体是专门设计用来在哺乳细胞中进行高水平的稳定表达和瞬时表达。其中人巨细胞病毒早期启动子(CMV)在广泛的哺乳动物细胞中可以实现高表达。另外为筛选稳定细胞系提供了新霉素抗性基因,可用G418筛选。
pcDNA3.0 is 5.4 kb vectors designed for high-level stable and transient expression in mammalian hosts. High-level stable and non-replicative transient expression can be carried out in most mammalian cells. The vectors contain the following elements: Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells. Neomycin resistance gene for selection of stable cell lines.
pcDNA3.0 vector is designed for high-level, constitutive expression in a variety of mammalian cell lines. It contains a Geneticin selectable marker and a forward-orientation multiple cloning site. High-level stable and non-replicative transient expression can be carried out in most mammalian cells. The vectors contain the following elements: Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells Multiple cloning sites orientations to facilitate cloning .Neomycin resistance gene for selection of stable cell lines Episomal replication in cells lines that are latently infected with SV40 or that express the SV40 large T antigen.
特征片段:
CMV promoter: bases 209-863 T7 promoter: bases 864-882
Polylinker: bases 889-994 Sp6 promoter: bases 999-1016
BGH poly A: bases 1018-1249 SV40 promoter: bases 1790-2115
SV40 origin of replication: bases 1984-2069 Neomycin ORF: bases 2151-2945
SV40 poly A: bases 3000-3372 ColE1 origin: bases 3632-4305
Ampicillin ORF: bases 4450-5310
质粒图谱
pCDNA3.0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。
pCDNA3.0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。