Figure 5.

Purification and electroporation of 1-Mb chromosomes. (A) The split-chromosomes of RGF152 were purified with NucleoBond Xtra BAC kit, analysed by agarose gel electrophoresis, electroporated into HST08. A 127.5 ng aliquot and 4 × 51 ng aliquots of purified DNA (total 21 μg) were used for electrophoresis and electroporation, respectively. By using four vials of commercial electrocompetent cells of HST08, two positive colonies were obtained. The 0.84-Mb Chr^LR chromosome was purified from YST01, one of the Chr^LR-transformed HST08 strains. The agarose gel electrophoresis analyses were performed using 0.5% agarose, 0.5× TBE, at 60 V for 65 min. The DNA size markers used were Marker 3 (λ/HindIII + λ/EcoRI digest mixture) and a 530-kb chromosome purified in the same manner. (B) The 1-Mb split-chromosomes of HF053 and HF054 were purified and analysed in the same manner as in (A). (C) PFGE analysis of the Chr^Ter chromosomes of six strains of YST03, HST08 transformed with the Chr^Ter chromosome purified in (B).