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Electroporation of Yeast Artificial Chromosomes

igested with BamHI, thereby freeing the telomeres and resulting in a 14.4 kb linear DNA molecule. The restriction-digested DNA was microdialized (Millipore VS, 0.025 m) against TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) for 20 minutes, ethanol precipitated, and suspended in TE. The electroporation protocol used for our experiments was an adaptation of the one devised by Becker and Guarente.5 Exponentially growing cells were harvested by centrifugation, washed twice in water (4 C), once in 1 M sorbitol (4 C), and finally resuspended in a 2/3 pellet volume of 1 M sorbitol (4 C). A 40 l aliquot of cells was gently mixed with 1-5 l of DNA (0.1 g) just before electroporation. A Gene Pulser apparatus connected to the Pulse Controller accessory was used to pulse the sample in a chilled 0.2 cm cuvette, generating a pulse with a field strength of 7.5 kV/cm (1.5 kV), and a time constant (τ) of approximately 4.2 msec (25 F and 200 Ω). Ice cold 1 M sorbitol (1 ml) was added immediately after pulse delivery, and 150 l aliquots were plated on SD ura- plates. Transformants were visible after 3 days of incubation at 30 C.


Results and Discussion
Transformations with the small YAC have yielded approximately 700 transformants / g of DNA. Transformations with a supercoiled 5 kb plasmid yielded up to 1.6 x 104 transformants / g of DNA. A number of parameters were investigated in attempt to increase efficiencies. We found that 0.1 g of DNA in 1-5 l of TE or water was the optimum amount for 40 l of cells (5 x 108 cells). More DNA had an adverse effect on transformation efficiency and less DNA did not seem to saturate the reaction (Figure 3). Spermidine
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