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yudaoqian88至尊木蟲 (知名作家)
不良人
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[求助]
Dual-Luciferase Reporter Assay System盒子注射煙草以后如何定量
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promega，E1910，Dual-Luciferase Reporter Assay System做蛋白和核酸互作，多數(shù)用的是質(zhì)粒轉(zhuǎn)化原生質(zhì)體，也有將質(zhì)粒轉(zhuǎn)化農(nóng)桿菌，然后注射煙草，取樣后經(jīng)過液氮研磨后測的，但沒有找打具體的說明和操作。問題如下： 1）煙草取樣量有什么依據(jù)？ 2）加入裂解液后需要培養(yǎng)裂解一段時間嗎？ 3）上機前，需要離心取上清測試嗎？ |
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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 (煙草). (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樓2018-10-18 14:23:48
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