pGreen-Puro-pSIH1-H1-copGFP-T2A-Puro慢病毒干扰质粒

基本信息

别名:	pGreenPuro
启动子:	CMV
复制子:	pUC ori
终止子:	SV40 poly(A) signal
质粒分类:	病毒系列，慢病毒克隆载体
质粒大小:	7.8kb
原核抗性:	Amp
筛选标记:	Puro
克隆菌株:	Stbl3
培养条件:	37℃，有氧 LB
表达宿主:	哺乳细胞
诱导方式:	无须诱导，瞬时表达
5'测序引物:	CMV-F：CGCAAATGGGCGGTAGGCGTG
3'测序引物:	根据序列设计引物

质粒简介
    The pGreenPuro™ Vector is designed to express a single-stranded shRNA sequence with a fold-back stem-loop structure (also known as a “hairpin”) from a RNA polymerase III H1 promoter .The hairpin-type siRNA (shRNA) template oligonucleotides need to be cloned into unique BamHI/EcoRI sites located just downstream of an H1 promoter. The pGreenPuro™ vector needs to be linearized by restriction digest with BamHI and EcoRI, and purified to remove the stuffer fragment. When linearized, the vector contains two unique 5’ overhangs to facilitate directional cloning of shRNA template oligos with minimal self-ligation background . When the shRNA construct is expressed from constitutive H1 promoter and terminated with the TTTTT sequence, the shRNA transcript folds into the hairpin structure, which is recognized by the DICER enzyme, cleaved to form a functional ds siRNA and transferred to a RISC complex for selective digestion of complementary target mRNAs .Two PCR primers are designed for regions flanking the shRNA insert in order to provide a simple way for screening of plasmid clones for the 质粒图谱


pGreen-Puro-pSIH1-H1-copGFP-T2A-Puro慢病毒干扰质粒使用说明：
    
     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。
 
pGreen-Puro-pSIH1-H1-copGFP-T2A-Puro慢病毒干扰质粒注意事项：
 
      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。