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microRNA 靶基因验证实验 

      
       当前,microRNA对靶基因的功能研究十分热门。利用荧光素酶报告基因检测实验寻找或验证microRNA对靶点作用性是不可或缺的实验技术。权阳生物可为您提供microRNA报告基因载体构建服务,并可进一步为您提供荧光素酶报告基因检测实验服务。您只需要提供目的microRNA靶位点序 列,Transheep专业技术服务人员就可替您完成载体构建,为您节省大量的时间和精力。

       下图为microRNA-reporter载体表达框 架示意图,具有多克隆位点,方便插入目的靶点序列。psiCHECK-2载体用于终点法的细胞裂解物检测,包含萤火虫荧光素酶基因,使海肾荧光素酶的表达水平归一化,令该系统更可靠、重现性更好。使用psiCHECK-2载体时,由于是一个载体,无须通过共转染进行归一化。



        



权阳生物microRNA报告基因实验流程:


构 建miR-reporter载体(含突变靶位点的荧光素酶报告基因载体)与内参质粒、microRNA mimics或 microRNAnegativecontrol共转染293T或Hela细胞。共转染细胞24-72h后,进行双荧光素酶检测,进一步验确定目的 microRNA的靶基因。
   
1.microRNA靶点的预测

利用Targetscan,miRanda,PicTar软件得到目的microRNA的预测靶点序列。

2.获得microRNA靶点序列
 
根据提供的靶序列,合成两条互补的单链DNA模板?;蛘?,设计引物PCR扩增目的microRNA靶基因3’UTR序列,或直接合成。

3.microRNA靶序列克隆
 
将合成的两条单链退火成双链(具有粘性末端),或将PCR产物双酶切,后连入经过同样双酶切的miR-reporter载体中,连接产物转化大肠杆菌DH5α。

4.阳性克隆鉴定
 
挑选转化的菌落,PCR筛选含有插入片段的阳性克隆,测序验证克隆的序列和目的microRNA靶序列一致。

5.细胞转染准备
 
将含目的microRNA靶基因的报告基因载体、内参质粒和microRNA mimics或microRNA negativecontrol共转染293T或Hela细胞。

6.荧光素酶检测
 
共转染细胞24-72h后,进行双荧光素酶检测,确定目的microRNA的靶基因。



整理分析数据,得出结论:   

  • 提供有microRNA靶序列的报告质粒,以及内参和对照质粒各一份。
  • 甘油保存菌株各一支。
  • 附mir-reporter载体的说明书
  • 以及实验报告:
  • 包括载体构建过程,鉴定记录,测序报告,细胞转染,荧光检测。
     
方案 价格 备注
miRNA、靶基因预测 免费 如果用mimic需另加350元mimic合成费用
靶基因3‘UTR双荧光素酶野生型载体(‹2.5kb) 2000元
靶基因3‘UTR双荧光素酶突变型载体(一个作用位点) 2000元
miRNA表达载体或mimic与双荧光素酶载体共转染 5500元
双荧光素酶检测
共计 9500元
优惠价 8800元
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联系我们→客户或销售人员与您沟通→签订合同→支付预付款→执行项目→确认收货→支付尾款→售后服务 Transheep提供免费的技术咨询,任何关于产品或者技术问题,都可以联系我们们。 免费热线:400-0179890 服务邮箱:[email protected] 基因合成
亚克隆构建
3,UTR验证实验
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腺病毒包装
稳转株构建
 
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