Selection Problem

[Techqueen]

Hi All, I have a bit of a problem. I set up a kill curve using Geneticin on a cell line H295R (Adrenal Carcinoma). After 14 days even at the highest concentration my cells are happy! That is a concentration of 2000ug/ml which should most definately be lethal.

Any suggestions........

[admin]

Wow,

first welcome techqueen to the forums!

Secondly, I would check your concentrations again. Your cell line may be resistant to this however at 2mg/mL you should re-check your dilutions. concentrations and weighing of powder just in case.

What is your LD50 at this concentration?

Cheers

[Techqueen]

Hi, First I use a premade solution at 50mg/ml which I dilute to the desired concentration. It is recommended that the working range is between 200 and 800ug/ml and I have used the same solution for a gene targeting experiment on ES cells with no problems.

I suspect as you say the cell line is resistant to Geneticin although I do get a degree of death. In untransfected cells they should certainly die at the concentration I used.

Now I wish I had used another selection agent in my constructs........