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Old Yeast Cell Sorting 1. Grow 3-5 ml overnight culture in YPD. Next morning, measure OD600. Dilute into 50 ml YPD to
 

Summary: 80
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 - 30C 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 30C
overnight. Aim for OD600 < 0.5 in the morning.
7. Place cells on ice. Once chilled (<15C), spin in GSA rotor 5 minutes at 5000 rpm at 4C. 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 4C. Wash each cell pellet with 10 ml ice cold PBS.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine