( igested with BamHI thereby freeing the...)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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