How do you make puromycin stock solution?
The stock solution of puromycin (10 mg/mL) is diluted in RPMI 1640 supplemented with 10% heat-inactivated (56 °C, 30 min) fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin/streptomycin. A total of 15 µL is added to 10 mL of medium in tube A.
How is puromycin concentration calculated?
Prior to using the puromycin antibiotic, titrate the selection agent to determine the optimal concentration for target cell line. Use the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection.
How much puromycin do I add?
Puromycin antibiotic ensures effective positive selection of cells expressing the puromycin-N-acetyl- transferase (pac) gene. In mammalian cells, the recommended working concentration range for puromycin is 0.5 – 10 µg/ml.
Is puromycin soluble in water?
The product is soluble in water (50 mg/ml), yielding a clear, colorless to faint yellow solution. The stock solution may be passed through a 0.22 µm filter and stored in aliquots at –20 °C. It is also soluble in methanol (10 mg/ml). Store the product at –20 °C.
How do I select cells with puromycin?
You want your selection with puromycin to be done within a week. So you should select a concentration of puromycin that would kill all untransfected cells in one week or less and leave only the transfected cells that will live and be stably transfected.
How does puromycin inhibit protein synthesis?
Puromycin is a naturally occurring aminonucleoside antibiotic that inhibits protein synthesis by ribosome-catalyzed incorporation into the C-terminus of elongating nascent chains, blocking further extension and resulting in premature termination of translation.
When should I start puromycin selection?
The selection (addition of puromycin to the cell culture media) is usually started around 24 hr following transfection. You can also start the selection at 48 hr depending on the health of your transfected cells.
How does puromycin inhibit translation?
Is puromycin toxic to cells?
It is toxic to prokaryotic and eukaryotic cells. The recommended dose as a selection agent in cell cultures is within a range of 1-10 μg/ml, although it can be toxic to eukaryotic cells at concentrations as low as 1 μg/ml. Puromycin acts quickly and can kill up to 99% of nonresistant cells within 2 days.