Sack: Cell Media with Selection Agents (Blasticidin, Zeocin, Geneticin)

Sack and Yarov-Yarovoy Labs

Department of Physiology and Membrane Biology
University of California, Davis

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Cell Maintenance Media with Selection Agents (Zeocin, Blasticidin, Geneticin) F12 from Gibco, 10% FBS, 1% Penicillin/Streptomycin, 1/10,000 Blasticidin, 1/4,000 Zeocin, 1/500 Geneticin Blasticidin = 10mg/ml stock (pcDNA6) Zeocin = 100mg/ml stock (pcDNA4) Geneticin, G418, stock (pcDNA3, pIRES) Obtain a 50mL conical tube and label as: “Ham’s F-12 Media + 10% FBS + 1% Pen/Strep + 1ug/ml blast + 25ug/ml zeo + 100ug/ul gen” Include date made and initials Add 44.5ml of Ham’s F-12 Media to the labeled 50mL conical tube Add 5ml of FBS Add 500µl of Pen Strep Add 5µl of blasticidin (Final Concentration (1µg/ml)) Add 12.5µl of zeocin (Final Concentration (25µg/ml)) Add 100µl of geneticin (Final Concentration (100µg/ml)) Mix by pipetting Close the conical tube tightly and store in 4°C To make selection media for establishing new cell lines, use 10x more selection agent for final concentrations of: Add 50µl of blasticidin to 50ml of Ham’s F-12 Media + 10% FBS + 1% Pen/Strep media (Final Concentration (10µg/ml)) Add 125µl of zeocin to 50ml of Ham’s F-12 Media + 10% FBS + 1% Pen/Strep media (Final Concentration (250µg/ml)) Add 1000µl of geneticin to 50ml of Ham’s F-12 Media + 10% FBS + 1% Pen/Strep media (Final Concentration (1000µg/ml))