Article Title: Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation
Figure Lengend Snippet: Generation of synthetic α 21-I TetO and α 21-II LacO/Gal4 arrays. (A) Scheme of the pBAC11.32TW12.32GLII containing BAC and YAC cassettes, G418 resistance cassette and synthetic DNA: α 21-I TetO formed by high ordered repeats (HOR) monomers (green arrows) containing CENP-B boxes (blue) alternate with monomers containing TetO (yellow); α 21-II LacO/Gal4 formed by high ordered repeats (HOR) monomers (yellow arrows) containing Gal4 binding sequence (green) alternating with LacO (red). (B) Schematic of the assembly of the α 21-I TetO and α 21-II LacO/Gal4 arrays. (C, D) PFGE analysis of the nascent α 21-I TetO and α 21-II LacO/Gal4 arrays, cut with BamHI/NotI after each cycles of tandem ligation array amplification as described in Figure S2A (C) and Figure S2B (D). Expected sizes: α 21-I TetO 11-mer 1 copy (1.9 kb), 8 copies (15.2 kb), 32 copies (60.8 kb); α 21-II LacO/Gal4 12-mer 1 copy (2 kb), 8 copies (16 kb), 32 copies (64 kb). Plasmid vector is 2.9 kb, BAC vector is 7.1 kb. The asterisk (*) indicates the fragments that have been cloned into BAC vector (8 copies, 16 kb); red arrow in D indicates the size of the final pBAC11.32TW12.32GLII (∼120 kb) (m and M, markers).
Article Snippet: After each cloning step, the forming arrays were digested with BamHI and NotI restriction enzymes (NEB) and analysed on 1% agarose gel electrophoresis using 100 bp DNA Ladder, Quick-Load 1 kb Extend DNA ladder or Low Range PFG Marker (NEB).
Techniques: BAC Assay, Binding Assay, Sequencing, Ligation, Amplification, Plasmid Preparation, Clone Assay