shRNA stable transfection and colony formation assay

1 post / 0 new
eyes
eyes's picture
shRNA stable transfection and colony formation assay

Dear all,

I'm planning to perform a clonogenic assay in order to evaluate the effect as tumor suppressor of a gene I'm studying.

The idea is to transfect cells with shRNA to generate stable transfectant, and then using the for a clonogenic assay. But in order to save time I was wondering if I could transfect cells with shRNA, treat them with the proper antibiotic (should be g418) to kill those that were non transfected, and then detach by trypsin and plate them again at the correct concentration (I think 1500 per well in a mw6). Somebody could tell me if this could be a good alternative that avoid me for waiting about one month and half before having some results? If so after replacing should I grow them in a selective medium (with g418)in order to keep alive only those cells incorporating the plasmid in their genome?

In some protocols I showed people prefer to plate cell in a soft agar when cells do not form colonies when plated on plastic. Can somebody tell me if it is necessary to use a particular kind of agarose or the use of the normal agar for LB plating E coli could be good as well.

And in the caseof the soft agar method how is it possible to count cells within colonies. In many protocols you find that a colony is defined to consist of at least 50 cells. When cells growth as layer I think should be easy count them but how is it possible when the colonies are spheres?

Thanks in advance

eyes