Materials

  • DNA samples
  • Yeast strains

Protocol

Preparation of Competent Cells

Grow yeast cells at 30˚C in 10 ml YPD broth until mid-log phase (~5 x 10xx6 - 2 x 10xx7 cells/ml or OD600 of 0.8-1.0). The following steps are accomplished at room temperature.

Usually, we dilute the fully grown culture 1:10, and let it grow for 4-6 hours to reach mid-log phase.

!IMPORTANT 
Aliquot amount of cells needed for further steps and change volumes accordingly for each reagent.
  1. Pellet the cells at 500 x g for 4 minutes and discard the supernatant.
  2. Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.
  3. 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. It is important to freeze the cells slowly. To accomplish this, either wrap the aliquotted cells in 2-6 layers of paper towels or place in a Styrofoam box before placing in the freezer. DO NOT use liquid nitrogen to snap-freeze the cells.

Transformation

This part of the procedure is the same for both frozen stored (thawed at room temperature) and freshly prepared competent yeast cells.

  1. To each well of a PCR plate, add 20 µl of competent cells with 0.2-1 µg DNA (2 µl volume).
  2. Add 200 µl of EZ 3 solution and mix thoroughly by pippetteing.
  3. Incubate at 30˚C for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2-3 times during this incubation.
  4. Spread 50-150 µl (or spot part of it) of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading.

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

  • interactome/yeast96tf.txt
  • Last modified: 2019/12/11 10:09
  • by dan