pGADT7-Rec2酵母单杂交质粒

基本信息

启动子:	T7,ADH1
复制子:	pUC ori
质粒分类:	酵母系列，酵母单杂交载体
质粒大小:	7.6kb
原核抗性:	Amp
筛选标记:	LEU2
克隆菌株:	DH5α
培养条件:	37℃，有氧 LB
表达宿主:	酵母细胞
5'测序引物:	T7:TAATACGACTCACTATAGGG
3'测序引物:	根据序列设计引物

质粒简介

     pGADT7-Rec2 is a cloning vector that can be used in yeast to express a protein of interest as a GAL4 activation domain (GAL4 AD) fusion. Transcription starts with the constitutive ADH1 promoter (PADH1) and ends with the ADH1 termination signal (TADH1). The GAL4 AD sequence includes the SV40 nuclear localization signal (SV40 NLS; 1) so that fusions translocate to the yeast nucleus. GAL4 AD fusions also contain a hemagglutinin (HA) epitope tag. The T7 promoter in pGADT7-Rec2 can be used for in vitro transcription and translation of the HA-tagged fusion protein. It also provides a binding site for sequencing with the T7 Promoter Sequencing Primer.
     pGADT7-Rec2 is a shuttle vector; it can be maintained in both yeast and bacteria. It contains an autonomous replication sequence (ARS4) and a LEU2 nutritional marker for replication and selection in yeast (2, 3). A centromeric sequence (CEN6) is included to ensure proper segregation of the plasmid during cell division (2, 3). For propagation and selection in E. coli, the vector contains a pUC origin of replication (pUC ori) and an ampicillin resistance gene (Ampr).
     pGADT7-Rec2 is derived from pGADT7-Rec, a cloning vector used in the Matchmaker (Two-Hybrid) Library Construction & Screening Kit (Cat. No. 630445). It was constructed by replacing the 2μ ori in pGADT7-Rec with the ARS4 and CEN6 elements. The ARS and CEN elements ensure stable, low-copy propagation of the vector. Unlike pGADT7-Rec, which is able to replicate multiple times during the yeast cell cycle, pGADT7-Rec2, with its ARS and CEN elements, can replicate only once during the cell cycle, so its copy number is restricted. Low-copy plasmids such as pGADT7-Rec2 are preferred for one-hybrid screening because they generate fewer false positives. In the original Matchmaker One-Hybrid System, copy number was restricted not by using low-copy, autonomously replicating plasmids, but by integrating the reporter construct into the yeast genome. With the development of pGADT7-Rec2 (and pHIS2, a low-copy reporter vector), integration is no longer necessary because each plasmid, with its CEN and ARS elements, now behaves like a minichromosome, both mitotically and meiotically.

质粒图谱

pGADT7-Rec2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。
 
pGADT7-Rec2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。