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- Perform a kill curve with various concentrations of antibiotics to determine the amount of antibiotics needed to kill the cells after one or two weeks.
- Do transfection as normal, and then maintain the cells in a selection agent for at least two weeks.
- Change the media every few days with new medium containing a selection agent.
- After two weeks or more, a single colony of clones out of cells by diluting them so that there is less than 1 cell per well on average (using a 96-well plate).
- Wait a few weeks, and check that they have formed colonies under the microscope.
- Extending the desired clone cells, and testing for protein expression either by immunofluorescence or western blot.
- Select the clones that have low, medium, and high expression, and below freezing cell batches.
- Selection pressure should be maintained as much as possible.