Protocol 1: Harvesting cells

 

  1. Streak out strains from permanent stock. Wait 2-3 days until colonies form. (In case of slow-growing mutants, streak out mutant strains first, then streak out wild-type strains a day or so later.) Always start cultures from freshly streaked plates to avoid revertants.

  2. Pick 3 or 4 colonies from each strain to start 10 ml cultures. Grow up overnight at 30°C. If mutant strains grows considerably slower, start mutant strain culture first (morning) and wild-type strain in the evening.

  3. Determine the OD600 of all cultures. The set of 3-4 cultures from each individual strain should result in similar OD600's. If not, there is a problem with this strain: test phenotypes. If the small cultures result in consistent OD600's then proceed. In morning dilute 2 of the wild-type and 2 of the mutant 10 ml cultures into >200 ml of pre-warmed YPD (and pre-warmed flask) to an OD600 of 0.1 or 0.05. If the mutant strain grows considerably slower it is advisable to start the wild-type strain at a 2x lower density. Be sure all of the media used was made the same day. Incubate at 30°C for 4-8 hr until the OD600 ~0.5. (N.B. pairwise comparisons of expression profiles of identical strains grown to even only marginally different OD's show significant differences.)

    FOR ts STRAINS ONLY
  4. Dilute 1:2 in 200 ml of pre-warmed YPD so that the temperature-shift temperature is correct (e.g., shifting to 37°C, pre-warm YPD to 44°C and prewarm final culture flasks and measuring flasks to 37°C). Use 500 ml flask to measure out correct volumes, then pour into 2 L flask and incubate at the appropriate temperature. Heat shock for exactly 45 minutes. Stagger heat shocking the individual cultures by at least 20 min.

    FOR ALL STRAINS
  5. Harvesting must take place as quickly as possible and identically for all strains whose expression profiles are to be compared. Differences in harvesting (pH water, length of time in centrifuge etc) will be reflected in differences in the expression profiles. For each culture: pour into 250 ml disposable centrifuge tubes (pre-cooled on ice). Remember to take an aliquot for determination of the OD600 before centrifugation. Centrifuge in GSA rotor 3 min at 3500 rpm (make sure adaptors are in place and rotor is precooled). Write down final volume and OD600 of cells.

  6. Immediately decant supernatant into sink. If not performed at once the pellet will not hold together (do not need to remove all liquid).

  7. Resuspend in 6 ml of ice cold ddH2O by pipetting up and down, transfer to next tube and continue. Pipette into 15 ml Falcon tube (pre-cooled on ice). Centrifuge in table top centrifuge 2.5 minutes at 2600 rpm.

  8. Aspirate off supernatant. Place in liquid nitrogen container (for at least 2 minutes). Store at -80°C. Steps 5-8 will take approximately 15 min depending on the acceleration and braking rates of the centrifuges. It is not advisable to harvest more than two cultures at a time.