What is the concentration of puromycin for selection?
0.5 – 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 select puromycin?
Puromycin Selection post transfection (shRNA)/transduction (lenti)
- 48 hours post-shRNA 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.
What is used for positive selection of recombinant ES cell?
Its basic design includes (1) a positive selectable marker, typically an antibiotic resistance gene expressed by a strong constitutive promoter, to select for embryonic stem cells that have incorporated the DNA sequence of interest, (2) a negative selectable marker, herpes simplex virus thymidine kinase (HSV‐tk) in …
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.
How do you make puromycin?
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.
What is the function of puromycin?
What is a positive clone?
The positive selection comes from a conditionally expressed lethal gene, such as a restriction enzyme, whose coding sequence contains the multiple cloning site. Any undigested or self-ligated vector copies retain a functional copy of the lethal gene and are therefore selected against in the transformation.
How is puromycin used in cell analysis?
Why is puromycin used in cell culture?
Puromycin is an antibiotic in which the researchers can use when working with cell cultures when they need a selective agent, a compound that will kill all cells without resistance genes, leaving only specific targets behind.
How is puromycin used in the selection process?
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. Puromycin can also be used in some particular conditions for the selection of E. coli transformants.
How long does it take to make Puromycin resistant cells?
Puromycin. 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.
How much puromycin do you need to kill a cell line?
The working concentrations of puromycin for mammalian cell lines range from 1 to 10 μg/ml. In a starting experiment we recommend to determine optimal concentrations of antibiotic required to kill your host cell line. Puromycin quickly kills eukaryotic cells that do not contain the pac gene.
How is Puromycin resistance conferred by the pac gene?
Resistance to puromycin is conferred by the puromycin N-acetyl-transferase gene ( pac) from Streptomyces. Puromycin has a fast mode of action, causing rapid cell death at low antibiotic concentrations.