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发布于:2018-12-27 12:13:31  访问:20 次 回复: 篇
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Was digested with EcoRI and EcoRV {and the|and also the
Was digested with EcoRI and EcoRV plus the PyycF-3 -myc-yycH fragment was subcloned into EcoRI and SmaI websites of vector pHT315 resulting in vector pJTF130. Considering the fact that the 3 -myc tagged fragment was not effectively detectable in western blots the c-myc-tag was expanded to 6 -myc. This was achieved by PCR GlyH-101Biological Activity amplification of pJTF129 with ON102 and ON103. The resulting fragment was digested with NheI and XbaI and ligated into the XbaI web site of pJTF130, resulting in pJTF131. This vector was transformed into the yycH-deleted strain JH25021 resulting in strain JH25077, which expresses a six -myc-tagged YycH protein. To construct a 6 7-tagged YycI expression plasmid, the yycI gene was PCR amplified from chromosomal DNA with ON104 and ON105. The amplified fragment was digested with XbaI and SacII and cloned in to the similar web-sites of vector pJTF116, resulting in pJTF118. The 3 7-tag was amplified by PCR on vector pSHU1 with ON106 and ON107 or ON108 and ON109. The two amplified fragments were digested with NdeI and BglII or BglII and XbaI, respectively and ligated with NdeI and XbaI digested pJTF118 inside a three-way ligation, resulting in vector pJTF132. The PyycF-6 7-yycI coding fragment was excised with PvuII and EcoRI and cloned into SmaI and EcoRI web-sites of vector pHT315, resulting in pJTF133. This vector was transformed into yycI deleted strain JH25022, resulting in strain JH25078, which expresses six 7 tagged YycI protein. Two hybrid plasmids Many of the two-hybrid plasmids utilized here have been PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/19373244 published previously (Daniel et al., 2006; Szurmant et al., 2007b). All other had been generated by amplifying the coding sequence for the respective genes introducing 5XbaI and 3KpnI web pages, and cloning the fragments into the vectors pKT25 and pUT18C. Bacterial two-hybrid experiments Protein interactions have been probed utilizing the bacterial two-hybrid assay of Ladant and colleagues (Karimova et al., 1998; Karimova et al., 2000). Within this assay, putative interaction partners are fused C-terminally either to Bordatella pertussis adenylate cyclase domain T18 or to domain T25. When interacting T18 fusion proteins and T25-fusion proteins are coexpressed inside the adenylate cyclase deficient E. coli strain BHT101, adenylate cyclase activity is reconstituted, which in turn leads to expression of -galactosidase. galactosidase activity was measured on X-gal indicator plates. Considering that a lot of on the constructs utilised here were toxic to the E. coli strain, this process is preferable to more quantitative liquid assays. The assay was performed as previously described (Daniel et al., 2006). Briefly, E. coli competent cells were co-transformed using the indicated plasmid combinations and plated straight as spots on an X-gal indicator plate, followed by development at 30 for 16 h. X-gal indicator plates had been composed of 1 Agar No. 1 (Oxoid), ten mM ammonium chloride, 1.2 mM ammonium nitrate, 1 mM magnesium sulfate, 0.75 mM sodium EGFR-IN-3MedChemExpress EGFR-IN-3 sulfite, 0.5 mM potassium dihydrogen phosphate, 0.1 mM manganese (II) chloride, and four M iron (III) chloride. The pH was adjusted to 7 with sodium hydroxide. Then, 0.8 glucose, 0.4 Casamino Acids, three M thiamine, 100 g of ampicillin/ml, 25 g of kanamycin/ml, and 0.004 X-Gal had been added for the medium before pouring the plates.Was digested with EcoRI and EcoRV along with the PyycF-3 -myc-yycH fragment
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