What is the concentration of puromycin for selection?

10 µg/ml
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.

How do you make a kill curve for antibiotics?

Dilute the cell suspension in complete medium and seed each well of 96-well plate so as that the final volume is 100 ul. The recommended cell density is ~50% on the day of treatment. Typical cell density before antibiotic treatment in a 24-well plate: 0.8 – 2.5 X 105 cells/ml of adherent cells.

How puromycin inhibits 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.

Does puromycin kill bacteria?

Puromycin is an aminonucleoside antibiotic produced by Streptomyces alboniger. It specifically inhibits peptidyl transfer on both prokaryotic and eukaryotic ribosomes. This antibiotic inhibits the growth of Gram positive bacteria and various animal and insect cells.

Why is puromycin not used to treat infections?

Puromycin is used in cell biology as a selective agent in cell culture systems. It is toxic to prokaryotic and eukaryotic cells.

Does puromycin affect eukaryotes?

How do you measure protein synthesis?

Protein synthesis rates can be measured in vivo by administering an amino acid as a tracer that is labeled with an isotope (radioactive or stable) of C, H, or N.

How to create a kill curve for puromycin?

Puromycin Kill Curve To create a Puromycin Kill Curve for your target cells, follow the protocol below. Aliquot cells in a 12-well plate, at a density such that they are at 72 hours from confluency. Add puromycin at concentrations of 0, 0.5, 1, 2, 5, and 10 μg/ml in six different wells.

What should the concentration of puromycin be in a cell?

In mammalian cells, the recommended working concentration range for puromycin is 0.5 – 10 µg/ml. Different cell types and cell culture conditions may require different concentrations of selection antibiotic.

What’s the lowest concentration of Puro for HEK 293?

We found that (over one week), the lowest concentration (0.25 ug/ml final) might be the best choice. Even 0.5 ug/ml was pretty toxic for the cells. Hi Sarah, you’re concentration sounds about right for many cell types. However, I’ve never used Puro for 293 myself, only various cancer cells.

How to calculate the kill curve of Cellecta?

Mix and place the cells at 37°C in a CO 2 incubator. Grow cells under standard conditions for 72 hours. Count viable cells, and determine the lowest concentration of drug that kills at least 95% of cells in 3-5 days.

What is the concentration of puromycin for selection? 10 µg/ml 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. How do you make a kill curve for antibiotics? Dilute the cell suspension in complete medium…