BAC-FISH Protocol for Chromosomal Analysis

DNA was isolated from single Escherichia coli colonies (QIAprep Spin Miniprep Kit; Qiagen) (Farrar and Donnison 2007 (link)), labeled with digoxygenin-11-dUTP and/or tetramethylrhodamine-5-dUTP (Roche Diagnostics) by nick translation, and used as molecular probes for BAC-FISH. In some cases, two or three BAC clones were simultaneously analyzed in various combinations (multi-BAC-FISH). These studies were carried out on mitotic metaphase chromosomes. Cytological preparations were made from root meristematic tissues, as previously described (Lesniewska et al. 2011 (link)). Slide quality was controlled by observation under a phase-contrast microscope (BX41; Olympus). FISH was performed according to the protocol previously adapted for use in L. angustifolius (Lesniewska et al. 2011 (link); Książkiewicz et al. 2013 (link)). Digoxygenated DNA probes were detected with FITC-conjugated antidigoxigenin primary antibodies (Roche Diagnostics). Chromosomes were counterstained with 2 μg/ml 4',6-diamidino-2-phenylindole (DAPI) (Sigma) in Vectashield antifade mounting medium (Vector Laboratories, Burlingame, CA). Preparations were examined under a BX 60 microscope (Olympus) using the Cell_F software (Olympus). The images were captured using a CCD monochromatic camera and superimposed using Micrografx Picture Publisher 8 software (Corel).