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Addgene: Virus Protocol - Generating Stable Cell Lines

(6 day ago) Some lentiviral vectors deliver mammalian antibiotic resistance (e.g., puromycin, blasticidin), which enables selection of a stable cell culture after transduction. Performing antibiotic selection on transduced cells enables elimination of untransduced cells, resulting in a more homogenous (but still polyclonal) cell population.


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Puromycin | Thermo Fisher Scientific - US

(8 day ago) Puromycin has a fast mode of action, causing rapid cell death at low antibiotic concentrations. 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.


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Optimization of Antibiotic Selection Using a Cytotoxicity

(12 day ago) If the concentration for the desired cell type is unknown, a titration experiment must be performed to determine the lowest concentration of antibiotic needed to efficiently select transduced cells. In this protocol, we highlight the use of puromycin, the antibiotic utilized with the standard pLKO.1 vector.


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Does anyone have a good protocol for puromycin selection

(5 day ago) Does anyone have a good protocol for puromycin selection for pSpCas9(BB)-2A--Puro transfectants? I agree with Matthew to do a Puromycin kill curve on your cell line if its different than HeLa


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Griffin:Puromycin Selection - OpenWetWare

(7 day ago) The working puromycin concentration for mammalian cell lines ranges from 1-10 µg/ml. Prior to using the puromycin antibiotic, titrate the selection agent to determine the optimal concentration for target cell line. Use the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection.


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Stable Transfection | Thermo Fisher Scientific - US

(6 day ago) Geneticin® (G418 sulfate), Zeocin™, hygromycin B, puromycin, and blasticidin antibiotics are the most commonly used selection antibiotics for stable cell transfection. These antibiotics provide unique solutions for your research needs, such as dual selection and rapid, stable cell line establishment. View all the Gibco® Selection Antibiotics


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A puromycin selectable cell line for the enrichment of

(28 day ago) Previously, 4 μg/mL puromycin was used for the Hb9-Puro cell line and 2 μg/mL was used here with the Sim1-Puro cell line since less than 100 cells/cm 2 were observed after 4 μg/mL puromycin selection (versus 50,000–100,000 cells/cm 2 for 2 μg/mL puromycin).


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Stable Cell Line Protocol - University of San Diego

(5 day ago) 3. To set up for the stable cell line selection, split cells and transfect with your construct. The following day, replace the standard media with media containing G418. Over time this will select for cells that have stably incorporated the GFP plasmid into their genomic DNA. The amount of G418 required to kill cells

http://home.sandiego.edu/~josephprovost/Stable Cell Line Protocol.pdf

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Generation of Stable Expression Mammalian Cell Lines Using

(7 day ago) Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1-2 weeks to get stable expression for most mammalian cell lines.


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Antibiotic Kill Curve | Sigma-Aldrich

(5 day ago) Table 1: Recommended Concentrations of Selection Antibiotics The dose response may vary with the cell line being tested. The concentration ranges are only for guidance; a kill curve must be performed with every selection antibiotic when used for the first time or used on a new cell line.


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