Frozen Yeast competent cells

http://www.nature.com/nprot/journal/v2/n1/full/nprot.2007.17.html

Points from here (point 1) up to and including point 9 are related to Cell growth

1. Cell growth
   Yeast strains not containing any plasmids should be grown in rich medium (option a). Yeast strains containing a plasmid that requires selection should be grown on selective medium (option b).
   1. Yeast cells without a plasmid
      Inoculate your yeast strain into 25 ml of liquid 2 × YPAD medium and incubate overnight on a rotary shaker at 200 r.p.m. and 30 °C. Place a bottle of 2 × YPAD and two sterile 2 liter or a sterile 4 liter culture flask with baffles in the incubator as well.
   2. Yeast strain with a plasmid
      Inoculate your yeast strain containing a plasmid into 200 ml of the appropriate SC selection medium in a 2 liter sterile flask and incubate overnight on a rotary shaker at 200 r.p.m. and 30 °C.
2. 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 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
      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.
3. 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^.
4. 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.
5. 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.
6. 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.
7. Dispense 50 μl samples into an appropriate number of 1.5 ml microcentrifuge tubes.
8. 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.
9. 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 1) and following are related to 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.

1. Incubate in a 42 °C water bath for 20--60 min depending on the strain. Temperature-sensitive strains can be left on the bench overnight and then carried on to the next step.
2. Centrifuge the tubes at 13,000g for 30 s in a microcentrifuge and remove the supernatant with a micropipettor. Transformations utilizing plasmids with prototrophic gene selection use option a and for those utilizing plasmids with eukaryotic antibiotic genes use option b.
   1. Prototrophic gene selection
      Pipette 1.0 ml of sterile water into the transformation tube. Stir the pellet with a sterile micropipette tip to break up the cell pellet and then vortex mix to thoroughly re-suspend pellet.
   2. Eukaryotic antibiotic gene selection
      Pipette 1.0 ml of YPAD liquid medium into the transformation tube. Vortex mix to thoroughly re-suspend pellet.
3. Incubate for 2--3 h at 30 °C to ensure good expression from the input plasmid DNA.
4. Plate 2, 20 or 200 μl of the cell suspension onto the appropriate SC selection medium. The 2 and 20 μl volumes should be delivered into a puddle of 100--200 μl of sterile water or YPAD depending on the selection (see Step 14). Once delivered, the inoculum is then spread with a glass rod, made sterile by being soaked in ethanol and passed through the flame of a Bunsen burner or alcohol lamp. The volume plated will depend on the efficiency of your yeast strain. Allow the liquid to be absorbed into the medium by incubation at room temperature. Cells should be plated less densely when possible, as plating density negatively affects transformation efficiency.
5. Incubate the plates at 30 °C for 3--4 days and recover the transformants.