产品详情
pLI50金黄色葡萄球菌单拷贝整合质粒
基本信息
复制子: Ori,repB
质粒大小: 5505bp
原核抗性: 氨苄青霉素Amp
筛选标记: 氯霉素Chl
克隆菌株: 大肠杆菌DH5α
培养条件: LB培养基,37℃,有氧
表达宿主: 金黄色葡萄球菌
5'测序引物: 根据全序列设计
3'测序引物: 根据全序列设计
质粒简介
pLI50质粒是一个金黄色葡萄球菌单拷贝整合载体,在大肠杆菌里氨苄抗性,在金黄色葡萄球菌里是氯霉素抗性。
Single-copy integration vectors suitable for cloning in Staphylococcus aureus have been constructed. Their construction was based on the site-specific recombination system of staphylococcal phage, L54a. The vectors are capable of autonomous replication in Escherichia coli, but they are not endowed with a replication function in S. aureus. As a consequence, establishment of these vectors in S. aureus can only be achieved by the integration system of the phage. Once integrated into the chromosome, the vectors, or their derivatives, were stably inherited even without selective pressure. Because such a vector exists in an integrated form in S. aureus, the gene dosage of the DNA cloned in the vector matches that of the chromosome.
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。