Team:Aachen/Wetlab/Protocol17

Making Competent Yeast Cells:


Material:


  • Frozen Competent Cell (FCC) Solution. FCC Solution is 5 % (v/v) glycerol and 10% (v/v) DMSO. Be sure to filter-sterilize.

  • Liquid YPD media. → about 1L

  • 50 mL Falcon.

  • styrofoam-box for freezer.

  • A 250 mL sterile culture flask for each strain.

  • A 2 L sterile flask for each strain.


Procedure:


  • First day:

  1. Inoculate 25ml of YPD with your strain (for an overnight cuture) and grow at 30 ℃ and 200 RPM overnight or for 12-16 hours.

  • Second day:

  1. Pre-warm 500 mL YPD in the 2 L flask at 30 ℃ for later use.

  2. .Take the overnight culture and find the OD600 to determine cell count (OD 0.1 = 1 x 10^6 cells/mL.) An OD close to 1 is desired. (dilute the culture with YPD-medium until you get an OD close to 1)

  3. With this culture, inoculate the pre-warmed flask of YPD with the entire 25mL and shake at 30°C for about 4 hours.

  4. centrifuge at 3,000xg for 5 minutes using 50 mL conical tubes. I split 250 mL of the culture among 5 tubes, spin them down, discard the supernatant, and add the remaining culture to the same tubes and repeat.

  5. Wash the cells in 0.5 volumes (25 mL in each of these tubes) of sterile water. Resuspend in 0.01 volumes (500μL each) sterile water and transfer to microfuge tubes. Pellet cells at 3,000xg for 5 minutes. Resuspend in 500 uL FCC solution and aliquot 50 uL each into microfuge tubes.

  6. Place these tubes into the styrofoam racks and store at -80 ℃. Since the racks are to promote slow freezing, after the tubes are brought to -80 ℃ you may transfer them to whatever your container of choice is in the -80 ℃.


Usage:


  • ake out a tube for each set of transformations and warm between your hands for 15 to 30 seconds. Pellet the cells at 3,000xg for 2 minutes and discard the supernatant. Take these cells and add them to your transformation master mix and follow the procedure as described in McClean:Yeast Transformation day 2 step 7. You may need to use more than one tube of cells if your yield was low.