Team:Aachen/Wetlab/Protocol20

Yeast Competence and Transformation Kit:

Preparation of competent yeast cells:


  1. Grow yeast cells at 30 ℃ in 10 ml YPD broth until mid-log phase (~5 x 10^6- 2 x 10^7cells/ml or OD600 of 0.8-1.0).

  2. Pellet the cells at 500 x g for 4 minutes and discard the supernatant.
  3. Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.

  4. Add 1 ml EZ 2 solution to resuspend the pellet.

At this point, the competent cells can be used for transformations directly or stored frozen at or below -70°C for future use.


Transformation:


  1. Mix 50 ul of competent cells with 0.2-1 µg DNA (in less than 5 ul volume); add 500 ul EZ 3 solution and mix thoroughly.

  2. Incubate at 30 ℃ for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2-3 times during this incubation.
  3. Spread 50-150 ul of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading.

  4. Incubate the plates at 30 ℃ for 2-4 days to allow for growth of transformants..