Puromycin dihydrochloride (CAS 58-58-2)

Puromycin is an aminonuclease antibiotic used for selection and maintenance of cell lines expressing a transfected pac gene, whose product, puromycin-N-acetyl-transferase, inactivates puromycin via acetylation.
This product has been cell culture tested and is recommended for selection of stably transfected cells following transfection with Santa Cruz Biotechnology shRNA or Lentiviral particles.
BACKGROUND: Puromycin is an antibiotic substance produced by the soil actinomycete Streptomyces alboniger which induces apoptosis in cells by interfering with RNA function, leading to inhibition of protein synthesis. Toxic to both eukaryotic and prokaryotic cells. Believed to act as an acyl-tRNA analogue causing premature chain termination, has been shown to arrest cells in G2/M phase.

For the selection of stably transfected cells following transfection with Santa Cruz Biotechnology shRNA or Lentiviral particles, proceed with puromycin selection as follows:
NOTE: The working puromycin concentration for selection in mammalian cell lines ranges from 1-10 µg/ml. Prior to using the puromycin antibiotic, titrate the selection agent to determine the optimal concentration for target cell line (see below). Use the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection.
• 48 hours post-transfection, aspirate the medium and replace with fresh medium containing puromycin at the appropriate concentration.
• Approximately every 2-3 days, aspirate and replace with freshly prepared selective media.
• Monitor the cells daily. Puromycin selection requires a minimum of 48 hours. Optimum effectiveness should be reached within 3-10 days.
• Assay transfected cells.
NOTE: Lot-to-lot variations in potency exist for all selection antibiotics, each new lot of puromycin should be titrated.
Titrating puromycin for selecting transfected cell lines:
• Plate 2 x 105 cells in each well of a 6-well plate containing 3 ml of the appropriate complete medium plus increasing concentrations of puromycin (i.e., 0, 1.0, 2.5, 5.0, 7.5, and 10.0 µg/ml)
• Replace with fresh selective medium after 2 days to remove dead cells.
• Examine the wells for viable cells every two days.
• Monitor the cells daily and observe the percentage of surviving cells. Optimum effectiveness should be reached in 1-4 days.
• The minimum antibiotic concentration to use is the lowest concentration that kills 100% of the cells in 3-5 days from the start of puromycin selection.

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