产品详情
pRI101-AN双子叶植物发根农杆菌质粒
基本信息
启动子: 35S
原核抗性: 卡那霉素Kan
筛选标记: 遗传霉素G418
克隆菌株: 大肠杆菌HB101
培养条件: 37℃
质粒简介
pRI101-AN载体是一种在双子叶植物细胞中表达外源基因的载体。利用发根土壤杆菌介导。pRI101 DNA vectors are binary plant transformation vectors intended for foreign gene expression in plant cells. These agrobacterium-mediated plant transformation vectors carry the 35S promoter of caμliflower mosaic virus (CaMV) and the 5’ non-coding region (5’-UTR) of the alcohol dehydrogenase (ADH) gene. The 5’-UTR of ADH functions as a translation enhancer in plants. There are two types of pRI101 DNA vectors, the pRI101-AN DNA vector, which carries the 5’-UTR of Arabidopsis ADH (AtADH 5’-UTR), and the pRI101 ON DNA vector, which carries the 5’-UTR of rice ADH (OsADH 5’-UTR). The pRI101-AN DNA vector is for dicotyledonous plants such as tobacco or Arabidopsis while the pRI101-ON DNA vector is for monocotyledonous plants such as rice.Although the pRI101 DNA vectors are shuttle vectors and replicate autonomously in E. coli and Rhizobium (Agrobacterium), they are high copy number plasmids because they contain the same replication origin as pUC-type plasmids (ColE1 ori). These vectors are also stably maintained in Rhizobium (Agrobacterium) containing the mutant-type replication origin Ri (Ri-ori). The pRI101 DNA vectors are capable of stably integrating target genes into plant chromosomes because the vector cloning sites are located closer to the Right Border (RB) of T-DNA than the selection marker (NPT II) of the plant, so the target gene is not deleted.
质粒图谱
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。