4 strains of E. coli are used in these studies: JM101 for M13 infection and isolation, XL1BMRF'for M13 or pUC-based DNA transformation, and ED8767 for cosmid DNA transformation. To maintain their respective F' episomes necessary for M13 viral infection, JM101 is streaked onto a M9 minimal media plate and XL1BMRF' is streaked onto an LB plate containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid.. - [Read Bacterial Cell Maintenance Protocol]
Stably transfected cells, generated in the first two stages of the procedure, are induced for expression of the target gene. After harvesting and lysis, the lysates are analyzed by SDS-PAGE and immunoblotting. - [Read Tetracycline as Regulator of Inducible Gene Expression III]
Protocol uses an autoregulatory system in which the transcriptional trans-activator tTA drives its own expression and that of a target gene. The first stage of the procedure describes how to generate stable lines of NIH-3T3 cells that express either tTA alone or tTA and the tetracycline-regulated target gene. - [Read Tetracycline as Regulator of Inducible Gene Expression Protocol]
This stage of the procedure describes the transfection with target genes of cell lines already expressing inducible tTA. In this example, the target genes are transfected on a plasmid that carries puromycin resistance as a selectable marker. - [Read Tetracycline as Regulator of Inducible Gene Expression Protocol II]