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question about G418 sceening test

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question about G418 sceening test

Postby cavaliersun2003 » Mon Nov 11, 2013 7:28 am

Hi, I'm quite new to cell culture experiment, so I'm looking for someone who can help me out here.
I'm trying to determine a threshold concentration for G418 screening of stable transfected cells (Huh-7). I start with a T25 flask of Huh-7 about 80% confluence, and passed it to a 6 well plate, each is about 15% confluence. After 24hr incubation with 2 mL regular DMEM, I changed to DMEM with G418 with different concentrations. The G418 disulfide 50mg/mL stock in H2O was from Sigma(G8168). I simply added it to 2mL to create a 400,500,600,700,800,900 ug/mL serie. It has been reported that 700 ug/mL was used for G418 screening for Huh-7. However, after 3 days incubation, all my cells in all wells were dead.
I have no idea why is that. One thing I noticed is that my Huh-7 grow really fast, it will take just 2-3 days to reach full confluency. By protocol I'm suppose to wait 10days to see the difference, so is that means the failure is somehow related to the fast growth rate of my cell? Any other comments?
Many thanks!

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