pESC-URA酿酒酵母质粒
基本信息
启动子: GAL1,GAL10 promoter
复制子: 2μ ori,ori,FI ori
终止子: CYC1
质粒大小: 6631bp
质粒标签: C-Flag, C-Myc
原核抗性: 氨苄青霉素Amp
筛选标记: URA3
克隆菌株: DH5α
培养条件: 37℃,有氧,LB
表达宿主: 酵母细胞
质粒简介
The pESC vectors are a series of epitope-tagging vectors for expression of eukaryotic genes in the yeast S. cerevisiae. Each vector contains GAL1 and GAL10 yeast promoters in opposing orientations. With these vectors you can introduce one or two cloned genes into a yeast host strain under the control of an inducible promoter. These vectors also feature an extensive polylinker sequence and the ability to generate end-specific RNA transcripts from T3 and T7 promoters. Each of the pESC vectors contains one of four different yeast-selectable markers (HIS3, TRP1, LEU2, or URA3) in the same vector backbone.
The pESC vectors contain DNA sequences coding for epitope peptides that can be specifically recognized by monoclonal antibodies. A sequence for the FLAG® epitope (DYKDDDDK) is located in the multiple cloning site (MCS) downstream of the GAL10 promoter; a sequence for the c-Myc epitope (EQKLISEEDL) is located in the MCS downstream of the GAL1 promoter. You can insert your gene of interest in front of the epitope sequence to generate C-terminal tagging or after the epitope sequence for N-terminal tagging. These tags allow the protein of interest to be studied without generating a specific antibody to that protein. The epitope-tagged fusion proteins can be studied in transformed cells using well-characterized antibodies.
• Express two different genes simultaneously in S. cerevisiae
• Proteins tagged with unique epitope tags
• Fast and easy immunoprecipitations
质粒图谱
![](http://shqshengbio.com/uploads/allimg/200826/1-200R6233U3252.png)
![](http://shqshengbio.com/uploads/allimg/200826/1-200R6233Z61E.png)
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。