Stable cell line - clone vs. population - (Feb/13/2008 )
take a stable transfected clone that reflects the properties of a stable transfected pool
i don't have much experience in working with cell culture but does one absolutely have to work with clones or would a population be ok also. i am trying to look at the end products following cleavage. i don't even think my lab has the right equipment to isolate clones.
we dilute a mix of stable clones to 1 cell/10µl, and seed 10 µl per well of a 96 well plate
i've read about "cloning cylinders". would i have to get one of these because my cells are adherent.
i thought these were need to isolate adherent cell clones. below is an example
You don't need cloning cylinders to isolate single clones. Serial dilution into a 96 well plate will do.
I used to cut a section of the flat end of a 200 ul pipet tip, autoclave it and spread it with some grease and press it onto indiviual colonies. This forms a sealed chamber. After removing the old medium, I add trypsin-EDTA to get cell suspension out of it. Obviously, you need to start with low cell density though. Some people just pick colonies right off the plate. 96 well plates works as well if you get the right concentration.
You mean cloning discs??
I find that works the best especially for difficult transfections where the expression of the protein significantly reduces the proliferation rate of the cell. I used about 8 96-well plates and I was able to get about 90 live colonies. After the screening process with ELISA to confirm protein expression, only 9 of the 90 was expression the protein of interest.