Hi, I want to select U266 which was transfected with plasmids contain Hygr gene that confers resistance to Hygromycin B, but I'm not sure this selection approach would work and have no idea about the optimal selection concentration. Is there anyone has done this experiment? Could you please give me some advices?
Hello Ting Jiang
Since cell lines differ in their sensitivity to antibiotics, the optimal concentration (pre-transfection) should be determined using an antibiotic kill curve. Antibiotic kill curve is a dose response experiment in which mammalian cells are subjected to increasing amounts of selection antibiotic to determine the minimum concentration of an antibiotic that can kill all the cells in a specific period of time.
Before experimentation, you should determine the optimal concentration of your antibiotic by developing the Kill Curve titration for your cells. You may follow the protocol provided in the link below for Hygromycin B. Once transfection has occurred, the cells can be treated to select for those cells expressing antibiotic resistance.
Please go through this link.
https://www.transomic.com/cms/Transomic/media/Homepage/FAQ%20Guidelines/CRISPR/FAQ-Kill-Curve.pdf
Best.
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