PiggyBac Dual promoter (PB513B-1)哺乳转座质粒

PiggyBac Dual promoter (PB513B-1)哺乳转座质粒

基本信息

别名: PB513B-1

启动子: CMV promoter

复制子: pUC ori,f1 ori

终止子: SV40 poly(A) signal

质粒分类: 哺乳系列质粒；哺乳编辑质粒；哺乳转座质粒

质粒大小: 7258bp

原核抗性: 氨苄青霉素Amp（100μg/ml）

筛选标记: 绿色荧光蛋白CopGFP、嘌呤霉素Puro(10ug/ml)

克隆菌株: DH5α等大肠杆菌

培养条件: 37℃，有氧 LB

表达宿主: 293T等哺乳细胞

培养条件: 37℃，5%CO2

5'测序引物: CMV-F（CGCAAATGGGCGGTAGGCGTG）

3'测序引物: Sv40-polyA-R（GAAATTTGTGATGCTATTGC）

质粒简介

        PiggyBac Dual promoter (PB513B-1)是用于哺乳细胞转座实验的质粒，位于CMV启动子下游的多克隆位点（MCS），使目的基因或microRNA更方便克隆。下游的EF-1α core promoter启动GFP的表达。

      The PiggyBac (PB) transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites. The powerful activity of the piggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes.

      The unique features of piggyBac transposons are that there is NO Cargo Limit and it is also Reversible. Genomes containing an inserted piggyBac vector can be transiently re-transfected with the PB transposase expression vector. The PB transposase will remove the transposons from the genome, footprint-free.The Super PiggyBac transposase transient expression vector and PB513B-1 were co-transfected into HeLa cells and puromycin selection applied for 10 days (10ug/ml). Cells efficiently transposed were Puro resistant and GFP positive.

质粒图谱

PiggyBac Dual promoter (PB513B-1)哺乳转座质粒使用说明：

     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。

PiggyBac Dual promoter (PB513B-1)哺乳转座质粒注意事项：

      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。