Koshland Web Site/Methods
Arresting Yeast Cells:

Alpha factor Arrest:

  1. Make a 10-3 M stock of alpha factor in 0.1 N HCl
  2. For BAR strains this is a 333X stock (final 3 X 10-6 M)
  3. For bar- strains this is a 66,600X stock (final 1.5 X 10-8 M)
  4. 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.

Hydroxurea Arrest:

  1. Make up 2M stock in H2O.
  2. This is a 10X stock (final 0.2M)

Nocodazole Arrest:

  1. Make up stock of 1.5 mg/ml in DMSO
  2. This is ~100X (see 4).
  3. Make culture 1% DMSO prior to adding Nocodazole
  4. Note different strains require different amounts of Nz. Too much Nz is bad and can induce break through the mitotic arrest. For best results a titration of Nz should be done to opitimize the best arrest conditions. Also breakthrough is more problematic at 37oC. In some cases it helps to add 50% more Nz a few hours into the arrest.
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