产品介绍
pABAI酵母单杂交质粒
基本信息
启动子: URA3 promoter
复制子: ColE1 ori
质粒分类: 酵母系列质粒;酵母杂交质粒;单杂交类质粒
质粒大小: 4894bp
原核抗性: 氨苄青霉素Amp(100μg/ml)
筛选标记: URA3
克隆菌株: DH5α等大肠杆菌
培养条件: 37℃,有氧,LB
表达宿主: Y1Hgold等酵母菌
培养条件: 30℃,YPDA,有氧
5'测序引物: pABAI-F(GTTCCTTATATGTAGCTTTCGACA)
3'测序引物: pABAI-R(CCATCTCGAAAAAGGGTTTGCC)
备注: 低拷贝质粒
质粒简介
pABAI质粒是一种单杂交酵母报告质粒,可以识别和分析蛋白质与DNA之间的相互作用,而不仅仅是证明蛋白之间的相互作用。这个质粒具有AbA抗性因而具有很强的筛选能力,可极大地降低背景水平。
pABAI is a yeast reporter vector that can be used in one-hybrid assays to identify and characterize DNA-binding proteins. The vector, which was specifically designed for use with the Matchmaker Gold Yeast One-Hybrid Library Screening System, contains a multiple cloning site upstream of the yeast iso-1-cytochrome C minimal promoter(not shown) and the AUR1-C gene, an antibiotic resistance gene that confers resistance to Aureobasidin A. A cis-acting element (i.e., a target sequence) can be inserted into the MCS and used as bait to screen GAL4 AD/cDNA fusion libraries for proteins that interact with the target sequence. Positive protein-DNA (i.e.,one-hybrid) interactions drive the expression of AUR1-C. As a result, one-hybrid interactions can be detected by selecting for yeast that are resistant to AbA.
pABAI cannot be propagated episomally in yeast; it can only be stablymaintained through integration into the host genome. Integration is accomplished via homologous recombination between the vector’s URA3 gene and the ura3-52 locus of the yeast strain provided in theMatchmaker Gold Yeast One-Hybrid System. URA3 is a nutritional marker that can also be used for selection of recombinant yeast. The vector also contains a Col E1 origin of replication and an ampicillin resistance gene (Ampr) for propagation and selection in E.coli.
To use pAbAi in a one-hybrid assay, clone one or more copies of a cis-acting element into the MCS. To facilitate recombination of the vector with the host’s ura3-52 locus, linearize the vector with either BbsI or BstBI, then introduce the linearized vector into competent yeast cells using the protocols in the Matchmaker Gold Yeast One-Hybrid Library Screening System User Manual.
Insertion of your target sequence may alter the basal expression of AUR1-C. Therefore, before starting a onehybrid analysis, basal levels of AUR1-C should be determined as described in the User Manual.
• Suitable host strains: DH5α and other general purpose strains.
• Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) to E. coli hosts.
• Copy number: low
质粒图谱
pABAI酵母单杂交质粒使用说明:
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。
pABAI酵母单杂交质粒注意事项:
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。