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1. After 12--16 h of growth, determine the titer of the yeast culture. This can be done using a spectrophotometer (option A) or a hemacytometer (option B).
   1. Using a spectrophotometer
      Pipette 10 μl of cells into 1.0 ml of water in a spectrophotometer cuvette, mix thoroughly by inversion and measure the OD at 600 nm (a suspension containing 1 × 10⁶ cells ml^−1^ will give an OD₆₀₀ of 0.1). Remember to multiply by the dilution factor to determine the titer in the cell culture.
   2. Using a hemacytometer
   3. Pipette 100 μl of suspension into 900 μl of sterile water in a microcentrifuge tube and mix thoroughly. Deliver 10 μl of this dilution onto the counting grid of an improved Neubauer hemacytometer, put the coverslip in place, wait several minutes for the cells to settle and count the number of cells in the 25 large grid squares using a microscope with a × 10 ocular and a × 10 objective lens. Multiply this number by 10,000 to obtain the titer in the diluted suspension. Remember to multiply by the dilution factor to determine the titer in the cell culture.
2. Add 2.5 × 10⁹ cells to 500 ml of the pre-warmed 2 × YPAD in the pre-warmed culture flask. The titer of this solution should be 5 × 10⁶ cells ml^−1^.
3. Incubate the flask in the shaking incubator at 30 °C and 200 r.p.m. until the cell titer is at least 2 × 10⁷ cells ml^−1^. This should take about 4 h.
4. Harvest the cells by centrifugation at 3,000g for 5 min, wash the cells in 0.5 volumes of sterile water, re-suspend in 0.01 volumes of sterile water, transfer to a suitable sterile centrifuge tube and pellet the cells at 3,000g for 5 min at 20 °C.
5. Re-suspend the cell pellet in 0.01 volumes of filter sterile frozen competent cell (FCC) solution (5% v/v glycerol, 10% v/v DMSO). Use good quality sterile DMSO.
6. Dispense 50 μl samples into an appropriate number of 1.5 ml microcentrifuge tubes.
7. Place microcentrifuge tubes into a 100 tube styrofoam rack with lid. It is best to place this container upright in a larger box (Styrofoam or cardboard) with additional insulation such as Styrofoam chips or newspaper to reduce the air space around the sample box. This will result in the samples freezing slowly, which is essential for good survival rates.
8. Put the large Styrofoam container in a −80 °C freezer overnight. The Styrofoam rack containing the frozen yeast cells can then be removed from the freezing container and stored at −80 °C*.{}

Points from here (point 10) up to and including point 16 are related to

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Cell transformation

1. Thaw cell samples in a 37 °C water bath for 15--30 s.
2. Centrifuge at 13,000g in a microcentrifuge for 2 min and remove the supernatant.
3. Make up frozen competent cell (FCC) transformation mix for the planned number of transformations plus one extra, according to the protocol described below. Include an extra tube for a negative control tube for no plasmid DNA. Add this to the pellet and vortex mix vigorously to re-suspend the cell pellet. Note the difference in PEG volume from all other procedures.

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