Could the strange G418 resistance fo my Chinese hamser cell lines be due to the - CAN I USE UNTREAED plates for selection? (May/14/2008 )
I am having a hard time with G418 resistance of my Chinese hamstr cell lines in a HOMOLOGOUS RECOMBINATION ASSAY. The cells are resistant even to a very high concentrations of g418. The conc. for selection is 1mg/ml in most of the papers that are performing this assay. Could the tissue culture treated dishes contain some detoxyfying protein that inactivates our G418 antibiotic? In the papers they have used 100 mm petri dishes. But they haven't said that they are tissue culture treaed or untreaed. Should i use the untreated ones for antibiotic selection?
-abyaseman-
QUOTE (abyaseman @ May 14 2008, 12:44 AM)
I am having a hard time with G418 resistance of my Chinese hamstr cell lines in a HOMOLOGOUS RECOMBINATION ASSAY. The cells are resistant even to a very high concentrations of g418. The conc. for selection is 1mg/ml in most of the papers that are performing this assay. Could the tissue culture treated dishes contain some detoxyfying protein that inactivates our G418 antibiotic? In the papers they have used 100 mm petri dishes. But they haven't said that they are tissue culture treaed or untreaed. Should i use the untreated ones for antibiotic selection?
You just need to wait more than a week! See the first result:
http://search.vadlo.com/b/q?sn=158621799&a...8+cho&rel=0
QUOTE
...CHO Lec 3.2.8.1 cells are relatively resistant to neomycin (GENETICIN G418; GIBCO #11811-031). It is better to draw kill curve for each batch of cells (and G418) every time you do transfection, but generally you can start with 1 mg/ml. Even with this high concentration, it will take more than a week to kill all of the non-transfected cells..
-cellcounter-
QUOTE (abyaseman @ May 14 2008, 10:44 AM)
I am having a hard time with G418 resistance of my Chinese hamstr cell lines in a HOMOLOGOUS RECOMBINATION ASSAY. The cells are resistant even to a very high concentrations of g418. The conc. for selection is 1mg/ml in most of the papers that are performing this assay. Could the tissue culture treated dishes contain some detoxyfying protein that inactivates our G418 antibiotic? In the papers they have used 100 mm petri dishes. But they haven't said that they are tissue culture treaed or untreaed. Should i use the untreated ones for antibiotic selection?
You need treated cell culture dishes, because they have a surface to which the cells can attach.
G418-selection is a bit difficult. Every new batch of G418 has a different active concentration.
-vista-