I am transfecting A375 cell line with pGFP-V-RS vector plasmid that carries puromycin mamalian marker.
I have done the killing curve for puromycin that was estimated to be 6 ug/ml of media.
so after 24 hours after confirming the GFP fluorescence, I started treating the cells with puromycin selection media. The problem is that number of cells with GFP are getting lessor and lessor with time. How to solve this problem
Thank you Sven.
When i did the killing curve for Puromycin:
cells treated with 2 ugm/ ml servived after 4 days of treatment.
cells treated with 4 ugm/ ml were 100% dead after 3 days.
cells treated with 6 ugm/ml and 8 ugm/ ml were 100% dead after 24hours.
so i used 6 ugm / ml which i do agree with you is high.
under the microscope i can still see few clones of viable cells. However when i try to detect the GFP fluorescence, none of the clones are visible.
I am going to do the transfection again and start Puromycin at 4 ugm/ml after 24 hrs and see the difference.
I hope the cells will survive this time
ces synthèses connotent l'entropie des regulations cellulaires en présence de nuisances
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