Kill Curve

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.





Procedure	Comments

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.