colon formation assay - (Mar/17/2010 )

Hi,

I nned to perform colony formation assay following transfection of my gene on interest (GOI). I have transfced my cells in a 6 well plate and I've heard its best to seed 2000 cells for the colony formation assay.

I have tried seeding 2000 cells and added the selection drug to the cells following 24 hours? Is this the normal practice?

Thanks

This is fairly standard practice. Some people seed less cells, down to about 500 or so per well, but it will depend on the growth rate of your cells. You may need to condition some medium and use that to keep the cells happy - 2000 per well is pretty low, and a lot of cell lines won't like it.

bob1 on Mar 18 2010, 07:25 AM said:

Hi,

when you say 2000 celss per well is quite low, do you mean its beeter to increase the number of cells/well?

Also, some one suggested 1:5 or 1:10 dilution of cells for seeding. I do not understand the mathematics very well. So, if I transfect my cells in 6 well plate,. 24 hrs following transfection , I trypsinise teh cells, centrifuge the supernatant and than add 5ml medium to make it a 1:5 dilution?

For a colony forming assay you want to form discrete colonies, so you need to have a low number of cells seeded. If you are doing this after transfection, I would recommend transfecting one well of a 6 well plate (or 35 mm dish) and then seeding all the cells from that out at 2000 cells per well (or whatever density of cells you decide to seed at)