Wittrup: Yeast storage and revival

Full-grown cultures can be stored at room temperature for ~2 weeks and at 4 degC for ~1 month. Frozen aliquots should be made for long-term storage (see below).

Yeast Library Freezing

Reagents:

Low-dextrose SDCAA (5 g/L dextrose, 6.7 g/L Difco yeast nitrogen base, 5 g/L bacto casamino acids, 5.4 g/L Na2HPO4, 8.56 g/L NaH2PO4-H2O)

Freezing solution (2% glycerol, 0.67% yeast nitrogen base)

Cryogenic vials

Isopropanol

1. Inoculate at least 10-fold oversampling of freshly grown cells into 1 L low-dextrose SDCAA and grow at 30 degC, 250 rpm for ~3 days.

2. Pellet cells and remove supernatant.

3. Resuspend the cells in freezing solution to give a final cell concentration of ~6x10^10/mL.

4. Aliquot at least 10-fold oversampling of cells per cryogenic vial. Incubate at room temperature for 10 min.

5. Place the vials in a bath of isopropanol at room temperature and slow-freeze the cells at -70 degC.

6. Transfer frozen yeast aliquots to liquid nitrogen for long-term storage.

Cell vials can be thawed at room temperature or 30 degC. Cells should be passaged after revival.