I want to optimize my cell transfection study using puromycin selection in A549 cells.
I have used 0.5, 1, 1.5, 2, 2.5 and 3 ug/ml of puromycin. I found out that all cells are killed in 1.5 and above puromycin. In 1ug/ml sample I see that more than 97% cells are died. I can actually count 100 cells which are alive out of 300000.
Should I use 1 or 1.5 ug/ml as a selection for my cells?
Regards,
Pratik
I want to use puromycin for resistance. I am trnafsectong to knock out a gene. The plasmid which knock out gene also introduced puromycin resistance genes.
Should I use 1 or 1.5 ug/ml. I was thinking 1
Because I can increase it to 1.5 anytime I want. The plasmid also has rpf gene so I should see florescence in cells once I see that many cells do not die after transfection.
Hi Pratik
Given that your plasmid has a visual marker (RFP)...so If you have invert floroscent microscope in your lab start the selection with a higher dose (for example 2 ug/mL) of Puromycin and check the viability of both the transfected and untransfected cells under microscope next days...note that once you found that high Puromycin doses kill a great number of the transfected cells in addition of the untransfected cells, so you should lower the dose.
Hi
I would say go for 1.5 ug/ml. Allow those few hundred to divide and then take those cells forward for your experiment.
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