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yudaoqian88至尊木虫 (知名作家)
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[求助]
Dual-Luciferase Reporter Assay System盒子注射烟草以后如何定量
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promega，E1910，Dual-Luciferase Reporter Assay System做蛋白和核酸互作，多数用的是质粒转化原生质体，也有将质粒转化农杆菌，然后注射烟草，取样后经过液氮研磨后测的，但没有找打具体的说明和操作。问题如下： 1）烟草取样量有什么依据？ 2）加入裂解液后需要培养裂解一段时间吗？ 3）上机前，需要离心取上清测试吗？ |
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xiaohongma
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2楼2018-03-05 07:28:27
yudaoqian88
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(1) The effector plasmid was constructed by cloning CDS into the vector. (2) Reporter plasmid, pGreen-LUC, encodes two luciferases: the firefly luciferase controlled by the recombinant E-box promoter and the Renilla luciferase controlled by the constitutive 35S promoter.. (3) pGreen-LUC reporter plasmid was transformed into Agrobacterium (strain AGL1) together with the helper lasmid pSoup-P19, which also encodes a repressor of cosuppression (Hellens et al., 2005). (4) Agrobacterium strain containing the reporter Green-LUC was used alone, or mixed with the Agrobacterium strain containing the effector plasmid. (5) Overnight cultures of Agrobacteria were collected by centrifugation, resuspended in the infiltration buffer (10 mM MES, 150 mM acetosyringone, and 10 mM MgCl2), and incubated at room temperature for 4 h before infiltration. The reporter strain was either incubated alone or as a mixture with the effector strain (at a reporter: effector ratio of 1:1). (6) Agrobacteria suspension in a 10-mL syringe (without the metal needle) was carefully press-infiltrated manually onto healthy leaves of 21-d-old N. benthamiana (烟草). (7) Plants were left under continuous white light for 3 d after infiltration, sprayed with luciferin (1 mM luciferin and 0.01% Triton X-100), and photographed using a charge-coupled device camera (Princeton Instruments). (8) Leaf samples were collected for the dualluciferase assay using a commercial kit (Promega; DLRreagents). Briefly, leaf discs infected with Agrobacteria were homogenized in 100 mL of passive lysis buffer. (9) Eight microliters of crude extract was mixed with 40 mL of LUC assay buffer, and the LUC activity was measured using a multimode microplate reader (Berthold; TriStar LB941). (10) Then, 40 mL of Stop and Glow buffer was added for the measurement of the REN activity. (11) Multiple biological repeats (n ≥ 3) were performed for each sample. |
3楼2018-10-18 14:23:48