Isolation of Yeast Nuclei
Growth of Yeast
Day -2 Grow a 5-50 ml overnight culture in YPD from a fresh patch of cells.
Day -1 Measure OD600 of the overnight culture. Dilute into 100-200 ml of YPD such that the
OD600 will be 0.5-1 at ~5 PM. Measure the growth rate during the day. For most strains,
the doubling time will be 110-120 minutes at 30°C.
Add starter culture to 1.5 L to give an OD600 = ~0.7 after the overnight growing period. Rotate at 150
rpm. Volume to add = [desired OD600/current OD600 X 2(# of doublings)] X final volume.
Preparation of Spheroplasts
1. Harvest yeast at OD600 = 0.6 - 0.8 (OD600 = 0.8 is ~5 X 107 cells/ml for strain BJ2168).
2. Centrifuge for 30 minutes at 4°C using 1 L bottles in IEC rotor at 2000 rpm. Or, centrifuge for 5
minutes in 250 ml bottles in Sorvall GSA rotor at 5000 rpm.
3. Resuspend cell pellets in ddH2O. Pool into 2 tared 250 ml centrifuge bottle(s). Centrifuge in
Sorvall GSA rotor for 5 minutes at 5000 rpm at 4°C.
4. Remove all of supernatant (decant and aspirate). Determine yield of cells (net weight of wet cell
pellet). Yield is typically ~50 g.
5. Resuspend cell pellets in ddH2O. Centrifuge again as in step 4. Prepare pretreatment buffer.
6. Pretreatment: Resuspend cell pellet(s) per 1 g of wet weight in 4 ml of freshly prepared
Pretreatment Buffer at room temperature. Swirl intermittently for 10 minutes at room