Does puromycin kill mammalian cells?
Does puromycin kill mammalian cells?
Mammalian cells Puromycin quickly kills eukaryotic cells that do not contain the pac gene. Dying cells detach form the plates allowing easy and early identification of transformant clones.
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 do you make puromycin solution?
How do you dissolve puromycin?
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).
How do you make a hygromycin stock solution?
For a stock solution of 5 mg/mL: 200 mg Hygromycin. 40 mL ddH2O. Filter sterilize.
What is Hygromycin B used for?
Hygromycin B has been used to select transfectants in a wide variety of cells including bacteria, protozoans, yeast, fungi, plants, and mammalian cells. Resistance to Hygromycin B is conferred by a gene coding for a phosphotransferase that phosphorylates Hygromycin B, thereby inactivating it.
How does puromycin interfere with 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.
What is the maximum concentration of puromycin that can harm mammalian cells?
Adherent mammalian cells are sensitive to concentrations of 2 to 5 µg/ml, while cells in suspension are sensitive to concentrations as low as 0.5 to 2 µg/ml. Puromycin-resistant stable mammalian cell lines can be generated in less than one week.
What temperature should puromycin be stored at?
Puromycin is shipped at room temperature. Upon receipt it should be stored at 4°C or -20°C. Puromycin is a harmful compound. Refer to safety data sheet for handling instructions. In a starting experiment we recommend to determine optimal concentrations of antibiotic required to kill your host cell line.
Can puromycin be used to select transformants?
Puromycin can also be used in some particular conditions for the selection of E. coli transformants. Resistance to puromycin is conferred by the Pac gene encoding a puromycin N-acetyl-transferase [1]. Animal cells are generally sensitive to concentrations from 1 to 10 µg/ml.
What is the pathophysiology of resistantresistance to puromycin?
Resistance to puromycin is conferred by the Pac gene encoding a puromycin N-acetyl-transferase. Animal cells are generally sensitive to concentrations from 1 to 10 µg/ml.
