I am using a stable clone which expresses my protein of interest, with G148 resistance and I use 500 ug/ml of G418 in the medium which is DMEM. Now I want to transfect with another plasmid having a cell cycle probe. Since this doesn't have any selectable marker, I am using puromycin which is in linear form.
First I plate the stable clone without G418 in the medium and the next day, I use 1 ug of DNA of the plasmid having cell cycle probe and 100ng of puromycin. I transfect with x tremegene 9, change the medium after 6 hrs and allow the cell to express for 48 hrs.
After two days, I change the medium which contains both G418 and puromycin (2ug/ml, conc determined by the kill curve). The next day all the cells died, which means that my experiment failed. what is want to know is
1. Why is this happening?
2. What is the rate of success when doing a experiment like this starting with a stable clone instead of using a normal cell line?
3. What should I do to get this successful.?
please help
HI, first check whether your transfection is working , which is very important!
For which you can co transfect with GFP plasmid.
Puromycin is a much more aggressive killer than G418. In my experience it is best to leave the transfected cells alone for two days and only then slowly start to add increasing concentrations of puromycin. I would definitely not hit the cells with 2 ug/ml from one day to another. After two days in no puromycin, I would add 250 ng/ml puromycin for a day. Next day, I would increase to 500 ng/ml puromycin. Next day, to 750 ng/ml puromycin. And then to a final concentration of 1.5 ug/ml puromycin.
Maybe try omitting the G418? You don't need that for anything, since your clone with the protein of interest is already stable, yes?
Your cell may be technically resistant to either antibiotic in isolation, but they still stress them out. When you hit them with both at the same time, plus the stress of transfection, I can imagine all that residual stress might conspire to kill them.
Thanks John. I thought that possibility, but if I dont add G418 I thought there might be a possibility of something growing in the culture, though I do the transfection with stable clone. May be next time I can try and later can add G418 when I get enough cells, so that if Iam going to loose some cells also, I can have a stable clone with the required result.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques