R.Jayakumar at roswellpark.org
Wed Nov 16 20:40:32 EST 2005
First estimate the kill curve for HEK293 for G418 and then do the
transfection with the amount of G418 that is toxic to the cell. You
shouldn't be estimating the selection dose of G418 after transfection.
The preliminary experiments for that should have been done before that.
Colonies should look normal with very little change in growth
period. Some cells may show a slightly longer doubling curve. Clone
the cells out and pick colonies that show normal morphology and normal
growth curve as that of control (no selection). A lot of things
decides how your cells behave after transfection including the site
where your construct has integrated into the nuclear genome unless
otherwise it is episomal (I have no idea what sort of expression you are
looking for).... But after 9 days, that should be stable transfection.
Picking up colonies is a whole different set of techniques. I use
both linear dilution technique in 96 well plates and cloning rings
(cloning rings available from Bell co). Pate around 200 cells per 96
well plate and that should yield you sinlge cell round colonies in at
least 10 % of the wells. Or plate them at 100 cells per 60 cm dish and
check for well separated round colonies after a week or so. Pick them
out using cloning rings. This is a bit tricky and needs practice.
Bye and best of luck
From: methods-bounces at oat.bio.indiana.edu
[mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of Raheleh
Sent: Tuesday, November 15, 2005 1:41 PM
To: methods at magpie.bio.indiana.edu
Subject: G418 selection
I have some monolayer HEK-293 transfected with Lipofectamin 2000 (
Invitrogen). 28 hours after transfection, I splitted the cells 1:8 and
the following day I added different concentrations ( 50 microgram/ml 400
and 800 microgram/ml) of G418 for selection. Ater 7 days I could see
there is difference between negative control ( transfected cells with no
G418) and other wells in terms of confluency. After 9 days I changed the
media and increased the amount o fG418 to 200, 500 and 1000
microgram/ml). Now I wonder how 293 colonies looks like. When I see the
colony, how should I pick it up?
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