Generating homologous mutant stem cells - any stricky point? (Jan/27/2007 )

Hi,

I am planning to generate homologous mutant stem cells. I have already heterozygous mutant stem cells that is generated by electroporation of a targeting vector and selected with low concentratio nof G418. I know that people can generate homologous mutant ES cells just by increaseing the G418 concentration up to 2 to 7 mg/ml. of the positive clone of heterozygous mutant ES cells.

However, here is the problem. ES cells are so sensitive. When G418 that is quite acidic, the medium colour changed immediately. And my colleague tried to this experiment before and all clone died in these concentration. My question, how do the people manage this problem? Do people need to fine tune the pH with KOH or NaOH after adding the G418?

thanks a lot.

I used up to 4.8 mg/ml G418 without adjusting the pH. Some cells survived and I did get some that were homozygous (although it wasn't the majority). The media did change color a little bit, but I checked with pH paper and it didn't seem too off.

The person I got the protocol from did say I may need to adjust the pH of the media, so I think that may be the way to go for you.

Good luck!
Julie

Thanks a lot, Julie

I will adjust the pH and to see whether it will help.

Ching