G418 Killing curve - How to determinate the right G418 concentration by killing curve ? (Mar/15/2007 )

Hi all, I have a little truble.
I'm tring to select stable trasfected cell line (THP-1) with G418 .
In order to determine the right concentration of G418 I'm doing a killing curve with non trasfected cells using a G418 concentration from 0.4 to 1mg .
My question is : how to determine the right concentration of G418 from the curve?
Is the concentration that kills the cells in the shortest time the one I have to choose?
Thanks for your help.
Raffa

-Raffa-

well you gonna have a concentration that will kill cells in a week
this one seems great to me.
G418 is not very quick at selection, so choosing quicker than 1week should give raise to too much toxcicity.

-fred_33-

QUOTE (Raffa @ Mar 15 2007, 06:22 PM)

I have best experience with low selection pressure (~ 350 µg/ml) initially for 12 days, then recovering, and an additional 5 days of selection; harsher selection which means higher conc of G418 may be faster but many selected cells refused to divide and were not usable for cloning

-The Bearer-

You need to select stbale tranfected cells. So you need a concentration which will kill all cells if they dont have the transfected plasmid.

G418 takes time, so wait atleast a week before you decide which conc. to use. if not sure, then wait longer or increase conc.

-scolix-

QUOTE (fred_33 @ Mar 15 2007, 01:17 PM)

You have to find out the minimum concentration that kills all your not resistan cells in one week.

-melisa-

QUOTE (melisa @ Mar 23 2007, 07:06 PM)

QUOTE (fred_33 @ Mar 15 2007, 01:17 PM)

You have to find out the minimum concentration that kills all your not resistan cells in one week.

-melisa-