产品详情
pFastBacDual昆虫双框质粒
基本信息
别名: pFastBac Dual
启动子: P10+PH promoter
复制子: pUC ori,F1 ori
终止子: SV40 poly(A) signal
质粒分类: 广宿主系列,昆虫细胞载体
质粒大小: 5238bp
原核抗性: 氨苄青霉素Amp
筛选标记: 庆大霉素Gen
克隆菌株: 大肠杆菌DH5α
培养条件: 37℃,有氧 LB
表达宿主: 昆虫细胞
诱导方式: 无须诱导,瞬时表达
5'测序引物: pFastbac-F: TATTCCGGATTATTCATACC
3'测序引物: pFastBac-R: ACAAATGTGGTATGGCTGA
质粒简介
pFastBac Dual昆虫细胞表达载体包含有两个启动子,能够同时表达两个外源基因,一个表达基因是polyhedrin (PH)启动子控制,另一个外源基因是由p10启动子控制。
pFastBacDual 载体的特点是在单个载体上有两个启动子,用于在使用 Bac-to-Bac® 杆状病毒表达系统(Bac-to-Bac® Baculovirus Expression System) 时在昆虫细胞中同时表达两个蛋白质。该载体具有两个强启动子,多角体蛋白启动子和p10 启动子,因此可实现高水平表达。包含 CAT和β-葡萄糖醛酸酶 (gus) 的表达对照。
The Bac-to-Bac® Vector Kit contains a pFastBac™ 1 vector, as well an expression control vector, intended for use as part of the Bac-to-Bac® Baculovirus Expression System , which enables the efficent production of recombinant baculovirus for expression testing in insect cells. The Bac-to-Bac® System relies on generation of recombinant baculovirus by site-specific transposition in E. colirather than homologous recombination in insect cells (Figure 1). This system features:
Time-saving expression bacmid. With Bac-to-Bac®, the expression cassette of the pFastBac™ vector recombines with the parent bacmid in DH10Bac™ E. coli Competent Cells (not included with this vector kit) to form an expression bacmid. The bacmid is then transfected into insect cells for production of recombinant baculovirus particles.
Easy colony screening. The parent bacmid in DH10Bac™ E. colicontains a segment of the lacZα gene. The lacZα gene is disrupted upon transposition of the expression cassette into the bacmid allowing for blue/white selection of recombinants. This makes identification of recombinant colonies easy.
质粒图谱
pFastBacDual昆虫双框质粒使用说明:
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。
pFastBacDual昆虫双框质粒注意事项:
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。