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Electroporation of Yeast Artificial Chromosomes

Maren Bell and Robert Mortimer, Human Genome Center, Division of Cell and Molecular Biology, Lawrence Berkeley Laboratory, Berkeley, California 94720


Introduction
Transformation of yeast with yeast artificial chromosomes (YACs) has traditionally been performed by a PEG-spheroplast procedure.1,2 However, the procedure is complicated, often unreliable, and transformation efficiencies are relatively low when compared to more recently developed transformation methods. An additional concern is that the smaller YACs of any given ligation mixture are cloned preferentially, therefore prior size fractionation with sucrose gradients to enrich for larger molecules, or treatment with spermidine to improve transformation efficiency of larger molecules, is required to obtain reasonable efficiencies with larger YACs. Several electroporation protocols have recently been reported for the transformation of Saccharomyces cerevisiae with supercoiled plasmids.3-6 In an attempt to improve the currently used YAC cloning procedure, we investigated the electroporation of S. cerevisiae strain AB1380 (Mat a, ade2-1, can1-100, lys2-1, trp1, ura3, his5, psi + ) with a 14.4 kb mini-YAC. Initial experiments showed transformation efficiencies two-fold better than those reported in a previously published YAC electroporation protocol.7


Materials and Methods
The YAC used in our experiments was constructed by the insertion of a 4.6 kb lambda MluI fragment into the MluI-SUP4 cloning site of pYAC-RC. 8 E. coli was transformed by the resulting 16.1 kb plasmid, the DNA was isolated, purified over cesium chloride, and restriction d
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