10 replies to this topic

[Evans]

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.

[bbryan]

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

[Buddy]

How did you resolve this problem. I too am having some trouble with my G418 selection assays and would appreciate if you could mention some of the things you changed to improve the situation.

Thanks,
Buddy

[chicken]

i worked on huh-7 cell lines .

before selection i checked the different concenration of g-418 (300 to 1000 ug/ml) for colony formation.and

i got 1000ug  as good selection concentration

[Dukey]

Just do a kill curve with various concentrations of G418 (without any transfection) and see what concentration is sufficient to kill all of the cells in, say, one or two weeks.

Edited by Dukey, 05 April 2010 - 12:54 PM.

[bob1]

Nice necro of an extremely old thread Buddy.  A kill curve is a good idea, you can also titrate the most effective concentration of plasmid to add.

[Buddy]

Thanks for the great advice. I did the kill curve under several conditions, and now I know the [] of g418 to use. I noticed that the process is more efficient if I change the media every day.

My lab is not very wealthy and g418 can get expensive. I was wondering if I could use less media. Currently, I am plating 10ml media on a 10cm plate. Could I go down to 7 or even 5 mLs(which barely covers the surface) if I am changing media that often.

Thanks again for the feedback,
Buddy

[Dukey]

Buddy, on Apr 16 2010, 08:36 AM, said:

Thanks for the great advice. I did the kill curve under several conditions, and now I know the [] of g418 to use. I noticed that the process is more efficient if I change the media every day.

My lab is not very wealthy and g418 can get expensive. I was wondering if I could use less media. Currently, I am plating 10ml media on a 10cm plate. Could I go down to 7 or even 5 mLs(which barely covers the surface) if I am changing media that often.

Thanks again for the feedback,
Buddy

Yeh that is a good observation, I definitely think you get better killing if you refresh media every day but I agree that G418 is VERY expensive when you are doing a lot of stable transfections. I use 10 mls on a 10 cm dish and wouldnt go lower than that for a very long time.

[Eric]

Buddy, on Apr 16 2010, 09:36 AM, said:

Thanks for the great advice. I did the kill curve under several conditions, and now I know the [] of g418 to use. I noticed that the process is more efficient if I change the media every day.

My lab is not very wealthy and g418 can get expensive. I was wondering if I could use less media. Currently, I am plating 10ml media on a 10cm plate. Could I go down to 7 or even 5 mLs(which barely covers the surface) if I am changing media that often.

Thanks again for the feedback,
Buddy

I would then even use higher concentration of G418. Beat them harder will definitely saves a lot than refreshing medium every day even use half of the medium as it may take some days for the non-transfected cells to die.

[izzy]

Hi,

I have been having trouble finding an appropriate selection concentration for G418 Sulfate on HCT116 cells. Its taken 2 weeks for the cells to die at a 1750 µg ⁄ ml concentration. Apparently they should die within 4-5 days at a maximum of 1000µg ⁄ mL concentration. Should I be doing something different? (besides typical things such as changing the media everyday, subculturing when overconfluent, applying the drug every day etc.)

Thanks!
izzy

[bob1]

There are two options - either your G418 has gone off (get a new batch to test and/or test on a known susceptible cell line), or you are not measuring death appropriately.  Try a MTT assay or something similar (even trypan blue) to assess death.