pRNAT-H1.1/Neo

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	Mouse mammary carcinoma cells 4T1, was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were incubated at 37°C with 5% (w/v) CO2 and 95% (w/v) air mixture.Two integrin α6 subunit oligos were designed by selecting appropriate sequences from the mouse integrin α6 gene (NM_008397) and named shRNA-1 and shRNA-control. The sequences of the two shRNAs were as follows:shRNA-: 5'-GATCCCCCCTTGTACACGGATTGAATTTCAAGAGAATT CAATCCGTGTACAAGGTTTTTA-3';shRNA-control: 5'-GATCCCCGGACAACGTGATCCGGAA ATTCAAGAGATTTCCGGATCACGTTGTCCTTTTTA-3'.The double strands for shRNA-1 and shRNA-control oligos were cloned into GeneScript shRNA expression vector, pRNAT-H1.1/Neo (Cat# SD1213-Genescript, NJ), using the BamH1 and HindIII restriction enzyme sites. The oligo insertion was confirmed by sequencing. After plasmid transfection, cells were cultured in G-418 (500 ug/ml) and several clones were isolated. The individual clones were characterized and positive clones were selected RT-PCR and Western blot analysis.

Protocol tips

Mouse mammary carcinoma cells 4T1, was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were incubated at 37°C with 5% (w/v) CO2 and 95% (w/v) air mixture.Two integrin α6 subunit oligos were designed by selecting appropriate sequences from the mouse integrin α6 gene (NM_008397) and named shRNA-1 and shRNA-control. The sequences of the two shRNAs were as follows:shRNA-: 5'-GATCCCCCCTTGTACACGGATTGAATTTCAAGAGAATT CAATCCGTGTACAAGGTTTTTA-3';shRNA-control: 5'-GATCCCCGGACAACGTGATCCGGAA ATTCAAGAGATTTCCGGATCACGTTGTCCTTTTTA-3'.The double strands for shRNA-1 and shRNA-control oligos were cloned into GeneScript shRNA expression vector, pRNAT-H1.1/Neo (Cat# SD1213-Genescript, NJ), using the BamH1 and HindIII restriction enzyme sites. The oligo insertion was confirmed by sequencing. After plasmid transfection, cells were cultured in G-418 (500 ug/ml) and several clones were isolated. The individual clones were characterized and positive clones were selected RT-PCR and Western blot analysis.

Publication protocol

Mouse mammary carcinoma cells 4T1, was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were incubated at 37°C with 5% (w/v) CO2 and 95% (w/v) air mixture.Two integrin α6 subunit oligos were designed by selecting appropriate sequences from the mouse integrin α6 gene (NM_008397) and named shRNA-1 and shRNA-control. The sequences of the two shRNAs were as follows:shRNA-: 5'-GATCCCCCCTTGTACACGGATTGAATTTCAAGAGAATT CAATCCGTGTACAAGGTTTTTA-3';shRNA-control: 5'-GATCCCCGGACAACGTGATCCGGAA ATTCAAGAGATTTCCGGATCACGTTGTCCTTTTTA-3'.The double strands for shRNA-1 and shRNA-control oligos were cloned into GeneScript shRNA expression vector, pRNAT-H1.1/Neo (Cat# SD1213-Genescript, NJ), using the BamH1 and HindIII restriction enzyme sites. The oligo insertion was confirmed by sequencing. After plasmid transfection, cells were cultured in G-418 (500 ug/ml) and several clones were isolated. The individual clones were characterized and positive clones were selected RT-PCR and Western blot analysis.

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