|- Use 20–25 ng of the target plasmid per 50 µL of mutagenesis reaction as a starting point.
|- Create a fresh dilution of 25X SAM from the kit-supplied 200X SAM in sterile, distilled water each time you perform the mutagenesis procedure
- 25X SAM is not stable, and loses activity within a few hours after
Mouse promoter, as shown in , was cloned into a luciferase-pcDNA3 plasmid upstream of the luciferase gene. The promoter/luciferase plasmid was mutated using the GeneArt Site-Directed Mutagenesis System (Invitrogen) at promoter E-box site (CATATG) (E3) by using two different sets of primers: Mutant 1 F: 5′- AGAGCTCATGTCTCTAGCTGCGGATGTAGCAGAA -3′, R: 5′- GCAGCTAGAGACATGAGCTCTGGGGGTACTGG -3′ and Mutant2 F: 5′- AGAGCTCATGTCTCTAGCTGCTGGTATAGCAGAAGAT -3′, R: 5′- GCAGCTAGAGACATGAGCTCTGGGGGTACTGG -3′. C2C12 cells were stably transfected with the wild-type and mutant /luciferase plasmids. Cells expressing the wild-type or mutant promoters were differentiated for 2 days before being transfected with AdT or AdC (control adenoviral vector) after which cells were differentiated for a further 2 days. Luciferase gene expression was then measured using Dual-Luciferase Reporter Assay (Promega). Cells were lysed and mixed with Luciferase Assay Substrate (LAR II) before measuring activity in a luminometer (Berthold). In order to ensure that luciferase readings reflect results originating from viable cells, the viability of cells in experiment was measured using a cell counter (Countess Automated cell counter, Invitrogen).
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