We are electroporating a pcDNA6.2 plasmid contain miR RNAi constructs into SH-SY5Y. I plan on selecting these cells with blasticidin, and the literature suggests closer to the 10ug/ml mark for this cell type. Ideally I would like to see effective selection by 96hrs of administration of blasticidin, in this case should I add more (maybe ~15ug/ml) or is there a degree to which efficiency tapers off or becomes excessively toxic to my cells?
Hi Martin,
1. In our lab before we make any stable cell lines we will perform a dose response on the parental cell line with desired antibiotic so that we know at what dose the parental cell line is completely dead then we use that dose to selecting out our transfected cells.
2. If you see 10 ug/ml blasticidin for your cell line in the literature. I will recommend go for it but make sure your uninfected cells are dying out in 3-4days if not immediately increase it 12-15 ug/ml.
In future before you do the transfection make sure that you follow the step to get a very clear stable cell population.
Good luck
Ram
Blasticidin is very potent antibiotic. 1-5ug/ml will work. Try to titrate the dose in parental cell line by using different concentration and see which dose is more toxic to cell line.
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