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 I select cells with puromycin?
You want your selection with puromycin to be done within a week. So you should select a concentration of puromycin that would kill all untransfected cells in one week or less and leave only the transfected cells that will live and be stably transfected.
How do you dilute puromycin?
If necessary, prepare an aliquot of 1mg/ml puromycin (working concentration) by diluting a 10mg/ml stock 1:10 with sterile water.
How does puromycin selection work?
This compound works by interrupting protein synthesis in both prokaryotic and eukaryotic cells. This causes cells to die, unless they have a gene for resistance, allowing them to survive exposure to the antibiotic.
How long does it take to select puromycin?
Puromycin selection requires a minimum of 48 hours. Optimum effectiveness should be reached within 3-10 days. Assay transfected cells.
What is the puromycin resistance gene?
Since puromycin is toxic to the growth of various eukaryote cells including mammalian cells, a puromycin-resistant gene (puromycin-N-acetyltransferase gene (pac)) is commonly used as a selection marker gene in mammalian cells (de la Luna et al., 1988, Vara et al., 1986).
How does puromycin inhibit translation?
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.
Is puromycin stable at 4 degrees?
Shipped at room temperature. Upon receipt it should be stored at 4°C. Puromycin is stable for up to three months at room temperature and at least one year at 4°C. For optimal stability and long term storage aqueous solutions can be stored at -20°C.
What is a molecular target of puromycin?
Puromycin is a ribosome-targeting antibiotic that mimics the aminoacyl-end of an aa-tRNA and quantitatively deacylates the P-site peptidyl-tRNA via peptidyl transfer; From: Methods in Enzymology, 2010.
How long is puromycin selection?
How long is puromycin stable?
Puromycin is stable for up to three months at room temperature and at least one year at 4°C. For optimal stability and long term storage aqueous solutions can be stored at -20°C.
When can I start selection after transfection?
Forty-eight hours after transfection, passage the cells at several different dilutions (e.g., 1:100, 1:500) in medium containing the appropriate selection drug. For effective selection, cells should be subconfluent, because confluent, non-growing cells are resistant to the effects of antibiotics like Geneticin®.
What is the final puromycin concentration for 293T cells?
Join ResearchGate to ask questions, get input, and advance your work. We use 0.4 ug/ml (final) of Puromycin for 293T, but for other cells it might be different. For example, I have done it for THP-1 (200,000 cells/well) and OCI-AML3 (150,000 cells/well), both in 12-well plate.
What is the best concentration of Puro for 293 cells?
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.
What is the best way to select 293T cells for transfection?
Do a dose test to see which lowest dose is capable of killing ALL untransfected 293T cells in 2-3 days and choose that dose. Use that dose for selecting transfected cells.
What is the recommended concentration of puromycin?
The recommended concentration of puromycin ranges from 1 to 10μg/ml according to cell lines. Additionally, the killing dose of puromycin vary from cell lines, 0.5-5 ug/mL in common.