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BioForum _ Molecular Cloning _ Time needed to determine G418 concentration for selection

Posted by: Daniel5306 Dec 13 2004, 09:48 AM

Hi,

I am trying to establish a stable transfection cell line. Before transfection, it is needed to determine a proper concentration of G418. As I know, a good concentration should kill all of the cell (non-transfected) within two weeks. Now I am trying the different concentration of G418 from 50-1ug/ml. The question is that how long time it is needed to determine the toxicity of the G418, if I have to wait two weeks?

Daniel

Posted by: mikew Dec 13 2004, 11:15 PM

IN general, G418 should kill all the cells in 4-5 days. At least, that has been the experience of the 3 labs I've been in. Usually a range of 500microgram - 1100 microgram /ml kills most cell lines within 5 days. Maybe 2 weeks works fine for you guys but you can be successful with 4-5 day toxicity and save alot of time. The clones usually become apparent within 2 weeks to 1 month.

Posted by: Daniel5306 Dec 14 2004, 08:40 AM

Hi, Mikew,

Thank your advice. I agree with you, two weeks is a little long for me. I will firstly try to kill the cell within one weeks and get a good concentration. Have a nice christmas!

Daniel

Posted by: iocxwl Sep 25 2009, 08:27 AM

Dear All:

I have set up a cell line 293, which was used for a stable transfection with pcDNA3.1/V5-HIS with a neomycin select gene. According to Invitrogen booklet, this antibiotics gene can be selected with Geneticin Selective reagent and a concentration of 100 to 1000 microgram/ml would be effective for the controll cells to die in 2-4 weeks. I used the Geneticin in 1000 microgram/ml concentration and now 5 weeks passed. Most of the control cells did not die even though last week I increased the concentration to 2000 microgram/ml. What is the problem? Can you give any advices on this matter?

Thank you very much.

Posted by: stardust Sep 26 2009, 04:49 AM

how confluent are your cells? G418 is not working properly if the cells are too dense. In this case I would advice one more passage directly into G418 containing medium.

Stardust

Posted by: Helios Nov 10 2009, 09:18 PM

according to me, you should have first checked the antibiotic concentration which is lethal to your cells by using different concentrations of your antibiotic and then checking your cell viability.......different cell lines have different susceptibility to the antibiotic....so what is recommended might not work well with your cells..you should standardise it yourself..