Preparation of Electrocompetent Cells
Yeast Transformation (Electroporation method)
For a Single Reaction:
- Innoculate a 5 ml culture of YPD with a yeast colony: Grow cerevisiae cells (BY4741). Prepare four 5 mL tubes of 1/10 serial dilution and incubate in a shaker at 225 rpm and 30 °C.
- The next morning pick a tube that is in the log phase (0.6~0.9).
- Harvest the cells by spinning at 3000 rpm for 5 min.
- At the spin rate of 4500 rpm for 5 min, wash the cell pellet twice with 5 mL ice-cold water and once with 5 mL of ice-cold electroporation buffer (1M D-sorbitol).
- Condition the yeast cells by resuspending the cell pellet in 5 mL 0.1 M LiAc/10 mM DTT and incubate in a shaker in an Erlenmeyer flask at 30 °C and 225 rpm.
- Collect the conditioned cells by centrifugation, wash once with 5 mL ice-cold electroporation buffer, and resuspended the cell pellet in 200 µL electroporation buffer.
- The cells are kept on ice until electroporation.
- Add DNA up to 1 µg. Do not exceed 5 µL for DNA.
- Gently mix 200 µL electrocompetent cells and DNA in a 1.5 mL microfuge tube and transfer the mixture to a pre-chilled BioRad GenePulser cuvette (0.2 cm electrode gap), using gel loading pipette tips. Keep the cuvette on ice for 5 minutes before performing electroporation.
- Electroporate the cells at 7kV. Typical time constant ranges from 3.0 to 6 milliseconds.
- Transfer electroporated cells from each cuvette into a 1.5 mL microfuge tube and add 1 mL of YPD. Do the recovery incubation in a shaker at 30 °C and 225 rpm for 1 hour.
- Spin cells down and resuspend in 150 µL electroporation buffer.
- Plate on selective media.