Puromycin Dihydrochloride Soluon
Quick Reference Protocol
Instrucons for MIR 5940
MSDS and Cercate of Analysis available at mirusbio.com/5940
SPECIFICATIONS
Mirus Bio LLC
Storage Store Puromycin Soluon at –20°C. Protect from moisture.
Product Guarantee As labeled on the product, when properly stored and handled.
Concentraon 10 mg/ml Puromycin Dihydrochloride, sterile ltered in DI water
Puromycin anbioc ensures eecve posive selecon of cells expressing the puromycin-N-acetyl-
transferase (pac) gene. In mammalian cells, the recommended working concentraon range for
puromycin is 0.5 – 10 µg/ml. Dierent cell types and cell culture condions may require dierent
concentraons of selecon anbioc. Perform a kill curve to determine the opmal working concentra-
on for your experiment. The following is a general guideline for performing an anbioc kill curve.
NOTE: Performing a kill curve is recommended with each new cell type or selecon anbioc lot, or if
changes are made to the cell culture condions.
A. Plate cells in 0.5 ml complete growth medium per well in a 24-well ssue culture plate.
For adherent cells: Plate cells at a density of 0.8—3.0 x 10
5
cells/ml.
For suspension cells: Plate cells at a density of 2.5—5.0 x 10
5
cells/ml.
B. Culture overnight. Most cell types should be ≥80% conuent prior to adding the selecon anbioc.
C. Add increasing amounts of puromycin to duplicate wells of cells plated in complete media. Include a
no-anbioc control. For example, add 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 µg/ml
Puromycin to duplicate wells of cells plated in complete growth media. Certain cell types and cell
culture condions may require concentraons outside of this range.
D. Replace media containing selecon anbioc every 2-3 days for up to a week. Examine the culture
every day for signs of visual toxicity. Determine the following anbioc doses:
- Low dose - the anbioc concentraon at which minimal visual toxicity is apparent aer 7 days of
anbioc selecon
- Opmal dose - the lowest anbioc concentraon at which all cells are dead aer 7 days of
anbioc selecon
- High dose - the anbioc concentraon at which visual toxicity is evident within the rst 2-3 days
of anbioc selecon
E. Proceed with stable cell line generaon using the concentraons determined in step D. Cells
transfected with a plasmid harboring the puromycin-N-acetyl-transferase (pac) gene should be grown
in complete growth medium for 48–72 hours post-transfecon before selecon anbioc is applied.
For more informaon on stable cell line generaon, visit www.mirusbio.com/stable.
ANTIBIOTIC KILL CURVE PROTOCOL
MSDS and Cercate of
Analysis available at
mirusbio.com/5940
