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yudaoqian88至尊木蟲 (知名作家)
不良人
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[交流]
pGREEN2-0800-LUC載體是否適合農(nóng)桿菌GV3101
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我們將pSOUP輔助質(zhì)粒轉(zhuǎn)入GV3101，長(zhǎng)斑倒是正常的，但無法在10mg/L正常濃度的四環(huán)素抗性液體培養(yǎng)基中上生長(zhǎng)，不知道是什么原因，一位做過這個(gè)實(shí)驗(yàn)的同學(xué)說需要共轉(zhuǎn)化。今天仔細(xì)看了文獻(xiàn)，指明可以共轉(zhuǎn)，也可以分兩步轉(zhuǎn)化。但在描述合適的菌株時(shí)未說明常用的GV3101能否使用，我們?cè)诓殚單墨I(xiàn)方法時(shí)，發(fā)現(xiàn)多數(shù)的研究者利用該載體也都是用的GV3101，特來求助。文獻(xiàn)摘取如下： 文獻(xiàn)信息如下： Plant Molecular Biology42:819–832,2000.©2000KluwerAcademicPublishers.PrintedintheNetherlands.819 pGreen: a versatile and fexible binary Ti vector for Agrobacterium-mediated plant transformation RogerP.Hellens,E.AnneEdwards,NicolaR.Leyland,SamanthaBeanandPhilipM.MullineauxJohnInnesCentre,NorwichResearchPark,Colney,Norwich,NR47UH,UK(authorforcorrespondence;e-mail:hellens@bbsrc.ac.uk;fax:C441603456844)Received8June1999;acceptedinrevisedform30January2000 Keywords: Agrobacterium ,binaryvectors, planttransformation, reportergenes, selectable markergenes, Ti vector 關(guān)于轉(zhuǎn)化部分的描述如下： The pGreen and pSoup plasmids (Figure1) can be used in a mixed electroporation. Inthisinstance, selection for co-transformed Agrobacterium can be achieved using kanamycin-containing medium only, since pGreen can not replicate in Agrobacterium without pSoup being co-resident. Alternatively, electro-competent Agrobacterium containing the pSoup can be generated, by selection for tetracycline resistance, and subsequently re-electroporated with pGreen and selection for kanamycin-resistant colonies: A.tumefaciens strains LBA4404, GV2260, GV3280, AGL-1 and EHA105 and A .rhizogenes strain LBA9402 support pGreen/pSoup replication. |
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chemg
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小木蟲: 金幣+0.5, 給個(gè)紅包，謝謝回帖
小木蟲: 金幣+0.5, 給個(gè)紅包，謝謝回帖
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請(qǐng)問psoup可以單轉(zhuǎn)大腸桿菌DH5α嗎？轉(zhuǎn)的話tet濃度是多少？ps:我們都是和pgreen共轉(zhuǎn)農(nóng)桿菌gv3101只加kana抗性，不加tet。希望能幫到你。 發(fā)自小木蟲Android客戶端 |
2樓2017-07-20 09:09:59
yudaoqian88
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3樓2017-07-20 10:43:00
qqhx
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4樓2018-04-23 16:59:31
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 (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. |
5樓2018-10-18 14:23:38
上官abc
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6樓2019-12-16 20:32:41
lc9031417
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7樓2021-09-09 08:55:20
yudaoqian88
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8樓2021-09-09 12:12:59
lc9031417
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9樓2021-09-15 21:09:56
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10樓2021-09-18 15:53:29
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