Team:Marburg/Protocols/Protocol25
Zymo Frozen Yeast Tranformation II Kit
Preparation of Competent CellsGrow yeast cells at 30°C in 10 ml YPD broth until mid-log phase (~5 x 106 - 2 x 107 cells/ml or OD600 of 0.8-1.0). The following steps are accomplished at room temperature.
- Pellet the cells at 500 x g for 4 minutes and discard the supernatant.
- Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.
- 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.
- Mix 50 µl of competent cells with 0.2-1 µg DNA (in less than 5 µl volume); add 500 µl EZ 3 solution and mix thoroughly.
- 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.
- Spread 50-150 µl 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.
Attention: For transformations of C. albicans, use freshly prepared competent cells; frozen cells sometimes give poor results.
