Before doing a transfection experiment, it is important to determine the concentration of selection reagent required for efficient selection. This is usually achieved by doing a kill curve, which is basically growing cells in various concentrations of the selection reagent. An important point to note when doing a kill curve is that each well must be seeded with a limiting number of cells, so that cell growth does not reach saturation in a short time and thus can be accurately quantitated. We can measure cell growth by measuring the no. of cells or the protein content, though I usually just measure the number of colonies on the plate after a specific time period.
|Prepare the selection reagent in a series of concentrations, making sure to include 0. Aliquot the various concentrations into as 24 well plate (each concentration in triplicate).||The 24 well plate is almost the size of the 10 x objective lens field, making it easier to count colonies.|
|Seed 100 cells into each well and gently mix.|
|Incubate for the required time, and then count the number of colonies as compared to control. Plot a graph if necessary. The least concentration which results in no colonies is a suitable level of selection.|