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钦诚生物 | 一周
3500¥
pAdVAntage哺乳辅助质粒
基本信息
启动子: Lac
复制子: pUC ori
质粒分类: 哺乳细胞,蛋白过表达载体
质粒大小: 4392bp
原核抗性: Amp
克隆菌株: DH5α
培养条件: 37℃,有氧 LB
表达宿主: 哺乳细胞
诱导方式: 无须诱导,瞬时表达
5'测序引物: M13R:CAGGAAACAGCTATGACC
3'测序引物: 根据序列设计引物
质粒简介
The use of pAdVAntage vectors were transfected into mammalian cells can promote the initiation of translation to enhance the instantaneous protein expression in many cell types.
Transfection of mammalian cells with expression vector often leads to the expression of the target protein. The double stranded RNA (dsRNA), which is produced in the transfection process, is thought to activate the dsRNA- activation inhibitor (DAI), which is one of the many kinds of enzymes involved in the host cell antiviral defense system. DAI phosphorylation of translation initiation factor eIF-2, termination of translation and generation of protein. However, with the pAdVAntage vector were transfected into DAI in the process of translation inhibition by RNA polymerase III generated adeno-associated virus type I (RNA VAI RNA) offset. RNA DAI combined with VAI to prevent its activation, so as to complete the translation and protein expression.
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。