Help! Problem with stable transfection of HCT116 - (Mar/07/2003 )
I have some problem with stable transfection using HCT116 cell line. I succeeded in transient transfection and western blotting proved my results. however, when I did stable transfection, I got so many colonies even I used 1000 ug/ml G418 (It seems this cell line is G418-resistent, however, no published paper confirmed this. ), so I have to split cells at a very big ratio (however, some experienced people said I shouldn't have got so many ones.). The western blotting didn't find interested band in the right postion. My target gene inserts into pcDNA3.1/His/C plasmids.
So my questions are:
1, Does anybody conduct stable transfection with HCT116? if yes, how much G418 was used in your case?
2, Is it possible that this cell line is contaminated? or G418 is out of function?
'cause I think G418 should kill all cells after weeks' culture. I just don't understand why I got so many colonies even I add hundreds of cells into a 10-cm dish.
Thank you for any suggestions and answers.
Evans -- I am also struggling to transfect HCT116, and I was wondering if you could help me. I've used Lipofectamine and Calcium Phosphate to transfect an expression vector encoding the specific enzyme that I want, but I can see no activity after harvesting the transient transfection, and I don't think the transfection is working. What specific protocols did you use to transfect this cell line? Just to make sure, I am adding G418 at 1000 ug/ml working concentration to attempt to establish a stable cell line, but what have you discovered regarding G418 resistance? Thanks! Bo