2. The next day, dilute the overnight culture to an A600 of 0.15~0.2 in a volume of 50 mL YPD in a flask large enough to provide good aeration.(Starting volumes can be scaled up or down.)
3. Grow yeast to an A600 of 0.8~1.0 in a 30℃ shaking incubator. based on a generation time of 100~120 min, yeast should reach 0.8~1.0 in 4 to 5 h.
4. Centrifuge the culture at 500×g for 5 min at room temperature and pour off the supernatant.
5.Re-suspend the pellet in 9 mL of ice-cold BEDS solution [10mM bicine-NaOH, pH 8.3, 3%(v/v) ethylene glycol, 5%(v/v)(dimethyl sulfoxide)DMSO, and 1 M sorbitol] supplemented with 1 mL 1.0 M dithiothreitol (DTT). Note that various concentrations (0~200mM) of DTT were tested. But the amount used in this procedure (100mM) yielded the most tranformants.
6.Incubate the cell suspension for 5 min at 100 rpm in the 30℃ shaking incubator.
7.Centrifuge the culture again at 500×g for 5 min at room temperature and re-suspend the cells in 1 mL(0.02 volumes) of BEDS solution without DTT. We have also found transformation efficiency may be increased by suspending cells in smaller volumes(0.005-0.01 volumes) of BEDS solution.
8.The competent cells are now ready for transformation. Alternatively, freeze cells slowly in small aliquots at -80℃ by placing the aliquots inside a Styrofoam box. Competent cells can be stored for at least 6 months at this temperature.
9.Mix approximately 4μL(50~100 ng) of linearized plasmid DNA with 40μL of competent cells in electroporation cuvette. Incubate for 2 min on ice.
10. Electroporate samples using the following parameters:
(i)ECM*630 electroporator(BTX, San Diego. CA. USA). Cuvette gap, 2.0 mm, charging voltage, 1500 V, resistance, 200Ω, capacitance, 50μF.
(ii)Gene Pulser* li electroporator (Bio-Rad Laboratories, Hercules, CA, USA). Cuvette gap, 2.0 mm, charging voltage, 1500 V, resistance, 200Ω, capacitance, 25μF.
11. Immediately after electroporation, re-suspend samples in 1 mL cold 1.0 M sorbitol and then plate on selective media(YNB, 2% dextrose, 1.0 M sorbitol) for auxotrophic strains. Alternatively, if using zeocin-based plasmids, re-suspend sample in 0.5 mL 1.0 M sorbitol and 0.5 mL YPD, incubate in a 30℃ shaker for 1 h, and then plate on media containing increasing concentrations of zeocin(100, 250, 500, or 1000 μg/mL.) for the selection of multicopy integrants. Note that increased numbers of transformants can be achieved for both types of selectable markers by incubating the re-suspended cells in a 30℃ shaker for longer periods of time(1~3h). However, this is partly due to the replication of transformants.
