G418 selection

Mukherjee, Sumit via methods%40net.bio.net (by sumit.mukherjee from ttu.edu)
Tue Sep 11 10:21:14 EST 2012


Hi 

I am a graduate student at Texas Tech University. I found a link in the internet regarding g418 selection. I have been trying to generate an inducible knocked down NK cell line. The vector I am using has a G418 resistant gene as selection marker. 48 hrs. after transfection, I am adding 0.2mg/ml of G418 to the media. At this concentration the cells are growing really well. However when I am increasing the concentration to 0.25mg/ml they are all dyeing even though from literatures I got to know that the optimum G418 concentration is higher than 0.25mg/ml. Generally when before changing the media, I keep the culture flask vertically for a couple of hours, to allow the cells to settle down at the bottom of the flask and then suck of most of the media. Then I add fresh media with G418. I have a stock solution of G418 (1mg/ml). So, if I add a total of 10 ml of fresh media, I add 7.5 ml of normal media and 2.5 ml of G418 (1mg/ml) containing media to reach to a final concentration of 0.25 mg/ml. 

MY question is whether the G418has any additive effect? I mean even though I am taking most of the old media before adding the fresh one, still I leave about 7-8 ml in the flask. Now this media will contain G418 (even though at a lower concentration). Is it like that the residual G418 along wit the freshly added one will give an additive effect and thereby will increase the actual concentration (more than 0.25mg/ml)?

What would you increase the G418 concentration in the selection media?

I t would be great to hear from you.

Thanks
Sumit



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