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.
There is a paper published in the Journal" Nature Reviews: Genetics" may help you.
November 2002 Vol 3 No 11
DNA POOLING: A TOOL FOR LARGE-SCALE ASSOCIATION STUDIES
Pak Sham, Joel S. Bader, Ian Craig, Michael O'Donovan & Michael Owen
Anybody has experience with Cell counting of Strongly adherent cells? The cell line I have is HCT116, which is a strongly adherent one. I tried many times but failed. I always find the cells gather tightly which makes my precise cell counting very difficult. Even pipetting up and down can't disperse them thoroughly.
Thanks whoever replies to this topic.