Stable cell line - clone vs. population - (Feb/13/2008 )
QUOTE (qkchen @ Feb 20 2008, 02:09 PM)
QUOTE (The Bearer @ Feb 19 2008, 12:52 PM)
QUOTE (qkchen @ Feb 13 2008, 09:39 PM)
Hi guys, I'm trying to transfect PC12 cells with a gene with selection under G418. I've been reading some protocols except I'm confused whether I should use a clone of transfected cells or a population of transfected cells. Any advice would be much appreciated. Thanks very much.
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
-The Bearer-
QUOTE (qkchen @ Feb 21 2008, 01:33 PM)
QUOTE (scolix @ Feb 21 2008, 06:49 AM)
To pick single clones, you need a 10ul pipette tip. After transfection, you seed the cells on a 10cm plate in a very low density with selection media. After a week or so, you will find individual colonies. You need to pick it up with the pipette tip and then transfer to another cell culture dish and keep in selecting media for some more time. And this would be your single clone.
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
http://www.biocompare.com/itemdetails.asp?itemid=361160
-qkchen-
You don't need cloning cylinders to isolate single clones. Serial dilution into a 96 well plate will do.
-lotus-
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.
-genehunter-1-
QUOTE
i've read about "cloning cylinders.
You mean cloning discs??
-zek-
QUOTE (lotus @ Feb 22 2008, 04:49 PM)
You don't need cloning cylinders to isolate single clones. Serial dilution into a 96 well plate will do.
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.
-rubyeye-