Puromycin Solution (10mg/mL)

Puromycin is an aminoglycoside antibiotic produced by the fermentation and metabolism of Streptomyces alboniger. In the field of cell biology is widely used as a screening antibiotic in mammalian cell culture systems. For screening eukaryotic or prokaryotic polyclonal or monoclonal cells obtained by plasmid transfection, transformation, virus infestation and other methods that can express the pac gene (puror). It is characterized by rapid cellular action, typically killing 99% of cells that do not express the pac gene in two days. Puromycin is used not only for the screening of stable cell lines, but also for the maintenance of stable cell lines. In addition, puromycin can also be used to screen for E. coli strains, yeast strains, etc that can express the pac gene.

The product contains 10 mg/mL puromycin, 20 mM HEPES, pH 6.2-6.8@25°C. After filtering and removing bacteria, it can be directly used for cell culture screening. Commonly used working concentration is 0.5-10 μg/mL. However, the optimal working concentration needs to be determined by a pre-experimental dose-response curve.

Ship withdry ice; Store at-20℃ to avoid repeated freeze-thaw, valid for12 months.

1. Determination of the dose-response curve for puromycin (Take shRNA transfection or lentivirus infection as an example):

The effective screening concentration of puromycin is correlated with the cell type, growth state, cell density, cell metabolism and the cell cycle. In order to screen for stably expressed shRNA or infected viral cell lines, it is important to determine the lowest concentration of puromycin that kills untransfected/infected cells. For first-time use of cells, it is generally necessary to experimentally determine the appropriate dose response curve for your experimental system.

(1) Day 1: 24-well plates are inoculated with cells at a density of 5~8x104 cells/well, and inoculate sufficient wells for subsequent dose gradient experiments. Incubate overnight in a cell incubator.

(2) Day 2: Cells are replaced in the overnight culture with freshly prepared screening medium containing different concentrations of puromycin (e.g. 0, 1, 2.5, 5, 7.5, 10 μg/ml, etc.), and continue incubation in the cell incubator after replacing the medium.

(3) After day 3: Because puromycin acts rapidly on cells, it generally kills 99% of cells that do not express the pac gene within 2 days. Therefore, the observation of cell survival rate can be performed 1-2 days after the addition of puromycin to determine the minimum concentration of the drug that effectively kills normal cells. If the cells are more resistant, daily observation is required and the minimum concentration of puromycin is usually determined within 4-10 days.

2. Screening of mammalian stable expression cell lines:

Stable expression strains can be screened after transfection with plasmids containing the pac gene or infection with viruses containing the gene.

(1) After 48h of cell transfection or infection, the cells are cultured in fresh medium containing appropriate concentrations of puromycin, which is the treatment group.

Note: The antibiotic effect is most pronounced when the cells are in an active phase of division. The effectiveness of antibiotic production is significantly reduced when the cells are too dense, so it is best to have a cell density of no more than 25%. It is recommended to also make a control group of normal cells. After 48h of transfection or infection, cells can also be digested and re-inoculated if they are too dense. Cells can be newly inoculated and cultured overnight for puromycin screening.

(2) Every 2-3 days, change the medium containing puromycin.

(3) After 7 days of screening, 100% of the normal cells in the control group should be dead and the surviving cells in the treated group are those expressing the pac gene. The cells are then screened for polyclonal or monoclonal cells depending on the purpose of the experiment.

Note: Observe cell growth status daily. The screening of puromycin requires at least 24 h. The screening period for effective concentrations of puromycin is generally 2-10 days.

(4) After the cells can be grown stably, the concentration of puromycin can be halved for subsequent cultures. After obtaining a stably expressed cell line, it is generally recommended to continuously add puromycin and to change the fresh culture medium containing puromycin on 2-3 days.

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For Research Use Only!