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Efficient Cloning and Electro-transformation of Large Eukaryotic DNA Fragments

Contributed by John M. Sedivy, Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, CT 06510.


Introduction
Genes of higher eukaryotes can be hundreds of kilobases in size. The need to rapidly clone and manipulate DNA molecules in that range has become acute in conjunction with the Human Genome Project. Cosmid vectors are constrained to 4045 kb by the packaging limit of phage lambda. Recently, three systems have been developed that extend this range: yeast artificial chromosome (YAC)-based vectors (>500 kb limit),1 phage P1-based vectors (100 kb limit),2 and E. coli F factor-based vectors (limit as yet untested).35 YAC clones have proven very successful, but are difficult to manipulate due to the extreme fragility of large linear DNA molecules. Bacterial episomes, being supercoiled, dont suffer that constraint. Unfortunately, F factor vectors have been limited by the low efficiency of transformation of large DNA molecules into E. coli. Recently, through the use of electroporation, efficiencies of 1010 transformants/g DNA of small plasmids have been achieved. A systematic analysis of large DNA molecules has been reported,5 and is summarized here.


Materials and Methods
E. coli strain JS46 was grown at 37 C in LB broth; in mid-log phase (OD600=0.6) flasks were placed in ice water and chilled for 15 minutes. Cells were harvested, washed in ice-cold 1 mM HEPES, pH 7.0 (first wash 1x, second wash 0.5x of original culture volume), and resuspended at 1011 cells/ml in 10% glycerol. 40 l aliquots were flash-frozen in dry ice/ethanol and
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