ChIP again... - (Apr/27/2005 )

Hi,

I'm trying to do ChIP for the first time, using the Upstate kit. It says that you should use 1 x 10^6 cells. They suggest that an extra plate of cells should be included to be able to count the cells.
Ok, but then I have to assume that there as many cells in the other plate, which aren't always true I guess, but perhaps that isn't as important. But what do I do if there are too many cells in the plate? Can I do the first steps with all my cells and cross-link and so one and before the sonication only take the volume that is equivalent to the right number of cells in the plate that I have counted? How do you with experience keep your cell number constant? Any technical tips?

Thanks in advance!

Hi,

You treat cells in duplicate, one for cell counting and the other for ChIP. If you use 100 mm dishes, you will get more than 1x10^6 cells. Go ahead with 1% formaldehyde crosslinking, add 5ml PBS and collect the cells. Then add 200 ul SDS lysis bufer to each 1x10^6 cells. So this is the place you need to adjust the number of cells. Although you can do it after sonication, but different number of cells may result in varying sonication efficiency.