Old Yeast Cell Sorting
1. Grow 3-5 ml overnight culture in YPD. Next morning, measure OD600. Dilute into 50 ml YPD to
yield OD600 ~ 0.05. Grow at 25 - 30°C to yield OD600 < 0.5.
· Place EZ-link biotin at room temperature.
· Place avidin magnetic beads on rotator in cold room to disperse.
2. Collect 1 X 108 cells. Spin in IEC centrifuge 10 minutes at 2000 rpm. If convenient, collect 2 X
108 cells and keep one tube on ice as a back up for step 5. For yeast strains we use, 1 X 107
cells/ml = OD600 ~0.25 (haploid), or OD600 ~0.3 (diploid). Use hemocytometer to be sure.
3. Wash twice with sterile PBS, pH 7.5. Resuspend 1 X 108 washed cells in 0.5 ml PBS.
4. Dissolve 8 mg of EZ-link biotin in 0.5 ml PBS. Repipet to dissolve. Add 0.5 ml of EZ-link biotin to
0.5 ml cells (8 mg EZ-link per 1 X 108 cells). Place on rotator at room temperature for 15 minutes.
EZ-link biotin is sulfo-NHS-LC-biotin (Pierce #21335).
5. Microfuge at top speed for 15 seconds to collect cells. Wash with PBS 7 times to remove biotin.
6. Resuspend cells in 1 ml YPD. Transfer to 1 L YPD at room temperature. Shake at 25 30°C
overnight. Aim for OD600 < 0.5 in the morning.
7. Place cells on ice. Once chilled (<15°C), spin in GSA rotor 5 minutes at 5000 rpm at 4°C. Pour off
supernatant immediately after spin to ensure that cell pellet is tight.
8. Resuspend and pool cells with 20 ml ice cold PBS. Transfer to 2 X 15 ml centrifuge tubes.
9. Spin 10 minutes at 2000 rpm at 4°C. Wash each cell pellet with 10 ml ice cold PBS.