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宙斯1111木虫 (著名写手)
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[求助]
谁用过荧光素酶瞬时表达载体，万分感谢万能的虫友们 已有1人参与
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就是这两个载体： pGreenII 0800-LUC vector pGreenII 0029 62-SK vector 谁用过啊？求帮忙，必有重谢~ |
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9楼2022-07-06 19:24:47
yudaoqian88
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宙斯11113楼2016-12-04 12:00:54
yudaoqian88
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For promoter activity assay, the MaLBD promoter region was amplified by PCR using the specific primers listed in Supplementary Table S1. The PCR product was inserted into the pGreenII 0800-LUC double reporter vector (Hellens et al. 2005) at the HindIII and BamHI sites to fuse it with the Firefly luciferase (LUC) reporter gene (MaLBDs pro-LUC); a Renilla (REN) luciferase under the control of the 35S promoter at the same vector was used as a positive control. The construct CaMV35S-REN/MaLBDs pro-LUC was transformed into tobacco BY-2 protoplasts by polyethylene glycol (PEG) methods as described previously (Abel and Theologis 1994). The promoter activity is indicated by the ratio of LUC to REN. |
4楼2016-12-04 12:02:43
yudaoqian88
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Transient Assay in Protoplast A dual luciferase assay was performed in the transient assay. For transactivation analysis of MaLBDs, the coding sequence of MaLBDs without the stop codon was cloned into the reconstructed GAL4DBD vector as effector. The double reporter vector includes a GAL4-LUC and a internal control REN driven by the 35S promoter. GAL4-LUC contains five copies of GAL4-binding element and minimal TATA region of the 35S promoter of CaMV, and these sequences are located upstream of the LUC. For the assay of the binding activity of MaLBDs to the MaEXPs promoter, two pGreenII vectors, pGreenII 0800- LUC reporter vector and pGreenII 0029 62-SK effector vector, were used (Hellens et al. 2005). The MaEXPs promoter was cloned into pGreen 0800 at the HindIII and BamHI sites to fuse it with the LUC reporter gene (MaEXPs pro-LUC). MaLBDs were cloned into the pGreenII 62-SK vector. The pGreenII 0800-LUC vector carried a REN under the control of the 35S promoter as a positive control. |
5楼2016-12-04 12:03:13