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pFastBac-GST-N昆虫胞内质粒
基本信息
启动子: Polyhedrin
复制子: pUC ori,F1 ori
终止子: SV40 poly(A) signal
质粒分类: 昆虫细胞载体;杆状病毒组成型表达质粒
质粒大小: 5463bp
质粒标签: C-3C,C-GST
原核抗性: 氨苄青霉素Ampicillin
筛选标记: 庆大霉素Gentamicin
克隆菌株: DH5α
培养条件: 37℃,有氧,LB
表达宿主: 昆虫细胞
诱导方式: 组成型表达,无需诱导
5'测序引物: pFastbac-F: TATTCCGGATTATTCATACC
3'测序引物: pFastBac-R: ACAAATGTGGTATGGCTGA
备注: 可表达GST融合蛋白
质粒简介
The Bac-to-Bac® Baculovirus Expression System provides a rapid and efficient method to generate recombinant baculoviruses (Ciccarone et al., 1997). This method was developed by researchers at Monsanto, and is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid) propagated in E. coli (Luckow et al., 1993). The major components of the Bac-to-Bac® Baculovirus Expression System include:
• A choice of pFastBac™ donor plasmids that allow generation of an expression construct containing the gene of interest where expression of the gene of interest is controlled by a baculovirus-specific promoter.
• An E. coli host strain, DH10Bac™, that contains a baculovirus shuttle vector (bacmid) and a helper plasmid, and allows generation of a recombinant bacmid following transposition of the pFastBac expression construct. .
质粒图谱
pFastBac-GST-N昆虫胞内质粒使用说明:
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。
pFastBac-GST-N昆虫胞内质粒注意事项:
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。