Alpha factor stock is 10-3 M in 0.1N HCl (i.e. resuspend 5 mg alpha factor in 2.97 ml 0.1N HCl). To arrest BAR1 strains, the pheromone should be diluted to a final concentration of approximately 3 x 10–5 M, while bar1 strains require much lower pheromone concentrations (10-8M). To release cells from alpha factor arrest, spin down cells and wash twice (using a large volume, dependent on the size of the cell pellet anticipated) in media containing 0.1 mg/ml Pronase E (Sigma P-6911). I generally make a fresh10 mg/ml stock in media (i.e. YPD or YEP-raffinose) and then sterilize using a syringe filter unit. Filter sterilization is not necessary unless you anticipate prolonged incubations after release from alpha factor.
Alpha Factor Arrest and Release
Alpha factor stock is 10-3 M in 0.1N HCl (i.e. resuspend 5 mg alpha factor in 2.97 ml 0.1N HCl). To arrest BAR1 strains, the pheromone should be diluted to a final concentration of approximately 3 x 10–5 M, while bar1 strains require much lower pheromone concentrations (10-8M). To release cells from alpha factor arrest, spin down cells and wash twice (using a large volume, dependent on the size of the cell pellet anticipated) in media containing 0.1 mg/ml Pronase E (Sigma P-6911). I generally make a fresh10 mg/ml stock in media (i.e. YPD or YEP-raffinose) and then sterilize using a syringe filter unit. Filter sterilization is not necessary unless you anticipate prolonged incubations after release from alpha factor.
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